Team:Kyoto/Notebook
From 2012.igem.org
Caution
We devided our team into 2 groups in order to achieve Flower Fairy E.coli, Florigen and Secretion group. Florigen aims to confirm expression of florigen in E.coli, activation of R9 peptide, and function of FT made by E.coli. Secretion group aims to make a secretion system without cell death and confirm function of tatABCD and the other proteins.
August 2
Mutation of FT
by Sato
FT gene has two BioBrick restriction enzyme sites, EcoR1 and Pst1 which is next to each other.
So we tried to delete both at once by using two primers with mutation.
10xBufer | 2mM dNTPs | primer fwd | primer rev | template | polymerase | MilliQ | Total |
---|---|---|---|---|---|---|---|
5 | 5 | 1.5 | 1.5 | 0.5 | 1 | 35.5 | 50 |
94℃ 2min, (98℃ 10sec, 68℃ 4min)x4cycles, 4℃ Hold
PCR product | Dpn1 |
---|---|
50 | 2 |
product | MilliQ | Ligase | T4 Kinase | Total |
---|---|---|---|---|
2 | 7 | 5 | 1 | 15 |
16℃, 1h incubate
competent cell | DNA |
---|---|
20 | 2 |
Cells were stored on ice for 30min.
After 42℃ 60sec heat shock, cells were stored on ice for 2min.
Then cells were precultureed at 37℃ for 1hr, plated to Kanamycin plate.
August 13
Liquid culture
FT at 37°C, for overnight.
August 14
Miniprep of FT
by Sato, Takeuchi
The concentration was 81.5ng/uL
Restriction digestion and Electrophoresis
by Sato
To check wheter mutation was succeed, we did restriction enzyme digestion.
DNA(FT,80ng/uL) | 10xBuferH | EcoR1 | Pst1 | MilliQ | Total |
---|---|---|---|---|---|
5 | 2 | 1 | - | 12 | 20 |
5 | 2 | - | 1 | 12 | 20 |
37°C 2h incubate
We did electrophoresis of three FT plasmids, non-cutted, EcoR1 cutted, Pst1 cutted.
However, we couldn't get any bands (data not shown.)
Liquid culture
FT (4mL)
August 15
Miniprep of FT
by Sato, Takeuchi, Hyungcheol
We couldn't get enough concentration of plasmids.
Electrophoresis
by Sato
We retried electrophoresis of three samples same as yesterday.
However, we couldn't get any bands as well.
August 16
Transformation
by Takeuchi, Ota
Name | Well | Sample | Competent Cells | Total | Plate | Colony |
---|---|---|---|---|---|---|
FT | - | 1 µL | 10 | 11 | LB (Kan+) | × |
pSB1C3 | 1-3-A | 1 | 10 | 11 | LB (CP+) | ○ |
I719005 | 1-15-N | 1 | 10 | 11 | LB (Amp+) | ○ |
August 17
Transformation
by Takeuchi
Name | Well | Sample | Competent Cells | MilliQ | Total | Plate | Colony |
---|---|---|---|---|---|---|---|
FT | - | 2 | 2 | 18 | 22 | LB (Kan+) | × |
We found that mutation of FT was not successful.
August 20
We decided to do PCR using FT specific primers before mutation.
PCR of FT
by Sato
10xBufer | dNTPs | MgSO4 | primer fwd | primer rev | template | polymerase | MilliQ | Total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1 | 1 | 1(130ng/µL) | 1(KOD plus neo) | 33 | 50 |
94°C 2min, (98°C 10sec, 68°C 15sec)x30cycles, 4°C Hold
After the ethanol precipitation, we diluted in 30µL of MilliQ.
The concentration was 203ng/µL.
Electrophoresis
Lane | Name | length(bp) |
---|---|---|
1 | 1kb ladder | - |
2 | FT | 600 |
August 21
Restriction digestion
by Sato
DNA(FT,203ng/µL) | 10xBuferM | Xba11 | Pst1 | MilliQ | Total |
---|---|---|---|---|---|
10 | 4 | 1 | 1 | 24 | 40 |
37°C, 5h incubate
After the ethanol precipitation, we diluted in 30µL of MilliQ.
The concentration was 54,9ng/µL.
Ligation
by Sato
Vector | Insert | Ligation High Ver.2 | ||
---|---|---|---|---|
pSB1C3 | 1 | FT | 10 | 5.5 |
Liquid culture
T7 promoter, pSB1C3 (4mL)
August 22
Miniprep
by Sato
T7 promoter | pSB1C3 |
---|---|
85.3ng/µL | 82.93ng/µL |
August 23
Ethanol Precipitation
diluted in 20µL 79.3ng/µL
August 24
Restriction enzyme processing
T7 promoter(85.3ng/µL) | Spel | Pstl | buffer M | MiliQ | Total |
---|---|---|---|---|---|
10 | 1 | 1 | 2 | 6 | 20 |
->purifying column 33.4ng/µL(dissolution 40µL)
pSB1C3(82.9ng/µL) | Xbal | Spel | buffer M | MiliQ | Total |
---|---|---|---|---|---|
20 | 1 | 1 | 4 | 14 | 40 |
->gene clean2 39.9ng/µL(dissolution 40µL)
Ligation
FT(600bp, 79.3ng/µL) | pSB1C3(2000bp,39.9ng/µL) | Ligation High Ver.2 |
---|---|---|
3µL => 597fmol | 2µL => 60fmol | 2.5µL |
FT(600bp, 79.3ng/µL) | T7(2100bp,33.4ng/µL) | Ligation High Ver.2 |
---|---|---|
2.4µL => 478fmol | 2µL => 48fmol | 2.2µL |
=> 16℃,1hr incubate
August 27
Colony PCR
2X Quick Tag | VF2 | VR | MiliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
Lane1: 1kb ladder
Lane2~16: FT(pSB1C3) about 800bp
FT(TOPO) PCR(re)
buffer | dNTPs | MgSO4 | primer f | primer r | Template(130ng/µL) | KODplus neo | MilliQ | Total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1 | 1 | 1 | 1 | 33 | 50 |
94℃ | 98℃ | 68℃ | cycles |
---|---|---|---|
2min | 10sec | 15sec | 30 |
Liquid culture
by Nobeyama
FT 4ml
August 28
Mutation of FT (re)
inverse PCR
MilliQ | buffer | dNTP | primer f | primer r | FT(130ng/µL) | KODplus | Total |
---|---|---|---|---|---|---|---|
35.5 | 5 | 5 | 1.5 | 1.5 | 0.5 | 1 | 50 |
94℃ | 98℃ | 68℃ | cycles |
---|---|---|---|
2min | 10sec | 4min | 18 |
Lane1: 1kb ladder
Lane2: FT
Miniprep FT(TOPO)
158ng/µL
Tranformation
competent cell: 20
BBa.I746902 : 2
(plate 316f)
(GFP generator of pBAD/araC-mut3 GFP:6His-DT)
August 29
Mutaion of FT(re;re)
Inverse PCR
first
MilliQ | buffer | dNTP | primer f | primer r | FT(130ng/µL) | KODplus | Total |
---|---|---|---|---|---|---|---|
35.5 | 5 | 5 | 1.5 | 1.5 | 0.5 | 1 | 50 |
second
MilliQ | buffer | dNTP | primer f | primer r | FT(52ng/µL) | KODplus | Total |
---|---|---|---|---|---|---|---|
35 | 5 | 5 | 1.5 | 1.5 | 1 | 1 | 50 |
94℃ | 98℃ | 68℃ | cycles |
---|---|---|---|
2min | 10sec | 4min | 30 |
Lane1: 1kb ladder
Lane2: first Template 130ng/µL
Lane3: second Template 53ng/µL
first sample | Dpnl | total |
---|---|---|
45µL | 2µL | 47µL |
in 37℃, 1 hour
Self-Ligation
PCR products | MilliQ | Ligation High | T4 kinase | total |
---|---|---|---|---|
2µL | 7µL | 5µL | 1µL | 15µL |
in 16℃, 1.5hour incubate
Transformation
competent cell: 20
DNA : 2
Liquid culture(I746902): 3mL
August 30
Liquid culture(FT) 4mL x2
August 31
Miniprep(FT)
(1) 64.9ng/µL
(2) 52.6ng/µL
Restriction enzyme processing (Mutation checking)
FT(52.6ng/µL) | bufferH | E.coli | Pst1 | MilliQ | total |
---|---|---|---|---|---|
1µL | 5µL | 0.5µL | 0.5µL | 3.5µL | 10µL |
in 37℃,1.5hour
PCR(RBS primer)
buffer for KODplus neo | dNTPs | MgSO4 | primer f | primer r | Template(52.6ng/µL) | KODplus neo | MilliQ | Total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1 | 1 | 1 | 1 | 33 | 50 |
94℃ | 98℃ | 68℃ | cycles |
---|---|---|---|
2min | 10sec | 15sec | 30 |
Lane1: 1kb ladder
Lane2: FT
Lane3: FT(E.coli)
Lane4: FT(Pst1)
Lane5: FT PCR
PCR(re)
buffer | dNTPs | MgSO4 | primer f | primer r | Template | KOD neo | MilliQ | Total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1 | 1 | 1 | 1 | 33 | 50 |
94℃ | 98℃ | 68℃ | cycles |
---|---|---|---|
2min | 10sec | 15sec | 35 |
Lane1: 1kb ladder
Lane2: FT
PCR(re)
buffer | dNTPs | MgSO4 | primer f | primer r | Template | KOD neo | MilliQ | Total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 2 | 1 | 1 | 1 | 1 | 34 | 50 |
94℃ | 98℃ | 68℃ | cycles |
---|---|---|---|
2min | 10sec | 10sec | 30 |
September 2
PCR(re;re)
first
buffer | dNTPs | MgSO4 | primer f | primer r | Template(1ng/µL) | KODplus neo | MilliQ | Total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1 | 1 | 1 | 1 | 33 | 50 |
second
buffer | dNTPs | MgSO4 | primer f | primer r | Template(10ng/µL) | KODplus neo | MilliQ | Total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1 | 1 | 1 | 1 | 33 | 50 |
94℃ | 98℃ | 68℃ | cycles |
---|---|---|---|
2min | 10sec | 10sec | 25 |
Lane1: first Template 1ng
Lane2: second Template 10ng
Lane3: 1kb ladder
refine first Template => 132ng/µL
September 3
Restriction enzyme processing
FT(132ng/µL) | bufferM | EcoRI | SpeI | MilliQ | Total |
---|---|---|---|---|---|
10 | 2 | 1 | 1 | 6 | 20 |
37℃,overnight => refinement 31.6ng/µL (Elution 40µL)
Ligation
Insert(FT: 31.6ng/µL, 600bp ): 2µL = 26fmol
Vector(DT: 28.0ng/µL, 3300bp): 3µL = 240fmol
Ligation High ver.2 :2.5µL
=> 16℃, 2 hours
Transformation
competent cell | DNA | plate | colony |
---|---|---|---|
20µL | FT-DT 2µL | Amp | o |
20µL | pT7-6His:R9 2µL | Amp | o |
10µL | GFP generator 1µL | Amp | o |
September 4
Colony PCR
Quick Tag | VF2 | VR | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃ | 98℃ | 55℃ | 68℃ | cycles |
---|---|---|---|---|
2min | 30sec | 30sec | 1min | 25 |
Lane1: 1kb ladder
Lane2: FT-DT(about 700bp)
Liquid culture(4ml) 23:30 ~
GFP generator, FT-DT
September 5
Miniprep(FT-DT) by Sato
88.8ng/µL
Restriction enzyme processing
FT-DT(88.8ng/µL) | XbaI | PstI | bufferM | MiliiQ | total |
---|---|---|---|---|---|
20 | 1 | 1 | 4 | 14 | 40 |
at 37℃, 2 hours
Electrophoresis by Takeuchi
Restriction enzyme processingby Takeuchi
FT-DT(88.8ng/µL) | bufferM | XbaI/Spe | MiliiQ | total |
---|---|---|---|---|
4 | 1 | 0.5 | 4.5 | 10 |
FT-DT | bufferH | Pst/Eco | MiliiQ | total |
---|---|---|---|---|
4 | 1 | 0.5 | 4.5 | 10 |
at 37℃,1hour 10min
September 6
Western blotting(BBa,I746915)
Sample making
SOC(ampt) 50ml + pre culture 1ml x2
OD600 = 0.5~0.7 incubate at 37℃ (OD 0.642)
add IPTG final concentration is 1mM (negative control)
incubate at 37℃,4 hours
SDS-PAGE
Do spin down E.coli and make suspension put E.coli into 1mL 1x sample buffer
95℃,10min
electrophoresis at 500V, 30mA, 50min
Lane1: 10µL (IPTG -)
Lane2: 10µL (IPTG -)
Lane3: 10µL (IPTG +)
Lane4: 5µL (IPTG -)
Lane5: 5µL (IPTG +)
Lane6: 2µL (IPTG -)
Lane7: 2µL (IPTG +)
Blotting at 50V,100mA,30min
Put into blocking buffer and vibrating 30min
Incubate with Anti GFP(1/1000) 10mL at RT,1h
Washing with 10mL TBST (vibrating 10min x2)
Incubate with Anti-mouseAP(1/1000) 10mL at RT, 30min
Washing with 10mL TBST (vibrating 10min x3)
Put NBT,BCIP into dye buffer
September 9
Mutaion of FT
MilliQ | buffer | dNTP | primer f | primer r | FT(52ng/µL) | KODplus | Total |
---|---|---|---|---|---|---|---|
35 | 5 | 5 | 1.5 | 1.5 | 1 | 1 | 50 |
94℃ | 98℃ | 68℃ | cycles |
---|---|---|---|
2min | 10sec | 4min | 20 |
→ GeneClean II 32.6ng/µL
Dpn1 processing
TA buffer | DNA | Dpn1 | Total |
---|---|---|---|
3.3 | 31 | 2 | 36.3 |
Ligation
DNA | MiliiQ | Ligation high Ver.2 | T4 kinase | Total |
---|---|---|---|---|
2 | 7 | 5 | 1 | 15 |
Transformation
competent cell: 10
DNA : 1
September 10
Miniprep of FT
①116.9ng/µL
② 34.5ng/µL
Restriction enzyme processing(Mutation checking)
FT①/② | bufferHl | EcoRI | PstI | MiliQ | Total |
---|---|---|---|---|---|
4 | 1 | 0.5 | 0 | 4 | 10 |
4 | 1 | 0 | 0.5 | 4 | 10 |
PCR
Bufer | dNTPs | MgSO4 | primer(+/-RBS)fwd | primer(+/-RBS)rev | template①/② | KOD plus neo | MilliQ | Total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1 | 1 | 1 | 1 | 33 | 50 |
94℃ | 98℃ | 68℃ | cycles |
---|---|---|---|
2min | 10sec | 10sec | 25 |
->purifying column 17.4ng/µL
Restriction enzyme processing
FT(RBS+)32.6ng/µL | Xbal | Pstl | buffer M | BSA | Total |
---|---|---|---|---|---|
30 | 1 | 1 | 4 | 4 | 40 |
T7-His:R9(62.6ng/µL) | SpeI | Pstl | MilliQ | buffer M | Total |
---|---|---|---|---|---|
15 | 0.5 | 0.5 | 2 | 2 | 20 |
incubate at 37℃, 3hours
->purifying column 17.4ng/µL
September 11
Ligation
Vector(T7: 33.4ng/µL, 2100bp): 1µL = 24fmol
Insert(FT: 17.4ng/µL, 600bp ): 5µL = 217fmol
Ligation High ver.2 : 3µL
=> 16℃, 1 hours
Transformation
competent cell | DNA |
---|---|
20µL | T7-FT 2µL |
PCR(FT without RBS retry)
Bufer | dNTPs | MgSO4 | primer(-RBS)fwd | primer(-RBS)rev | template | KOD plus | MilliQ | Total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1 | 1 | 1 | 1 | 33 | 50 |
94℃ | 98℃ | 65℃ | 68℃ | cycles |
---|---|---|---|---|
2min | 15sec | 30sec | 30sec | 25 |
Lane1:FT(RBS-) 600bp
Lane2:Ladder 100bp
=>purifying column 34.6ng/µL
Restriction enzyme processing
FT(RBS-) | XbaI | PstI | bufferM | BSA | total |
---|---|---|---|---|---|
30 | 1 | 1 | 4 | 4 | 40 |
at 37℃, 2 hours
=> 6.2ng/μL
GFP-DT | XbaI | PstI | bufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
15 | 1 | 1 | 3 | 3 | 7 | 30 |
at 37℃, 2 hours
=> 7.0ng/μL
Ligation
Vector(T7-6His-R9: 11.6ng/µL, 2100bp): 1µL = 9fmol
Insert(FT: 6.2ng/µL, 600bp ): 5µL = 79fmol
Ligation High ver.2 : 3µL
=> 16℃, 1 hours
Vector(T7-6His-R9: 11.6ng/µL, 2100bp): 1µL = 9fmol
Insert(GFP-DT: 7.0ng/µL, 1000bp ): 9µL = 95fmol
Ligation High ver.2 : 4µL
=> 16℃, 1 hours
September 12
Transformation
competent cell | DNA | plate |
---|---|---|
20µL | T7-R9-GFP-DT 2µL | Amp |
20µL | T7-R9-FT 2µL | Amp |
BL21CDE3 10µL | T7-FT 1µL | Amp |
Miniprep (T7-FT)
37.1ng/µL
September 13
Miniprep
①T7-R9-GFP-DT 90ng/µL
②T7-R9-FT 160ng/μL
Restriction enzyme processing
T7-R9-GFP-DT (90ng/µL) | EcoRI | PstI | buffer H | MilliQ | total |
---|---|---|---|---|---|
20 | 1 | 1 | 3 | 5 | 30 |
at 37℃, 2 hours
=> 20.7ng/μL
T7-R9-FT (160ng/µL) | EcoRI | SpeI | buffer M | MilliQ | total |
---|---|---|---|---|---|
20 | 1 | 1 | 3 | 5 | 30 |
at 37℃, 2 hours
=> 20.1ng/μL
Transformation
competent cell BL21(DE3) | DNA | plate | |
---|---|---|---|
10µL | T7-R9-GFP-DT 1µL | Amp | |
10µL | T7-R9-FT 1µL | Amp |
Restriction enzyme processing
Buffer H | BSA | EcoRI | PstI | DpnⅠ | MilliQ | total |
---|---|---|---|---|---|---|
5 | 5 | 0.5 | 0.5 | 0.5 | 13.5 | 25 |
=>We define this solution "2× Master Mix"
2× Master Mix | pSB1C3(Linerarized Plasmid Backbone) |
---|---|
4 | 4 |
at 37℃, 30 minutes
80℃, 30 minutes
PCR(Insert His-tag)
MilliQ | Buffer for iPCR | dNTPs | primer fwd | primer rev | template(T7-FT 37.1ng/μL) | KOD plus | Total |
---|---|---|---|---|---|---|---|
35 | 5 | 5 | 1.5 | 1.5 | 1 | 1 | 50 |
94℃ | 94℃ | 56℃ | 68℃ | cycles |
---|---|---|---|---|
2min | 15sec | 30sec | 2.5min | 15 |
Lane1:Ladder 1kb
Lane2:T7-6His:FT about 2700bp
September 14
Ligation
Vector(pSB1C3: 12.5/µL, 2000bp) : 2µL = 19fmol
Insert(T7-R9-GFP-DT: 20.7ng/µL, 700bp ): 4µL = 180fmol
Ligation High ver.2 : 3µL
=> 16℃, 1 hours
Dpn1 processing
PCR products(9/13) | Dpn1 | Total |
---|---|---|
45 | 2 | 47 |
=>37℃, 1 hour
Self Ligation
PCR products | Ligation High | T4 Kinase | MilliQ | Total |
---|---|---|---|---|
2 | 5 | 1 | 7 | 15 |
=>16℃, 1 hour
Ligation
Vector(DT: 17.1/µL, 2100bp) : 2µL = 12fmol
Insert(T7-R9-FT: 20.1ng/µL, 650bp ): 5µL = 496fmol
Ligation High ver.2 : 3.5µL
=> 16℃, 1 hours
September 15
Restriction enzyme processing
FT without RBS (34.6ng/µL) | EcoRI | PstI | 10× buffer H | MilliQ | total |
---|---|---|---|---|---|
10 | 0.5 | 0.5 | 2 | 7 | 20 |
Colony PCR
Quick Tag | VF | VR | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃ | 94℃ | 55℃ | 68℃ | cycles |
---|---|---|---|---|
2min | 30sec | 30sec | 1min | 25 |
Electrophoresis
Lane1: ladder
Lane2: T7-R9-GFP-DT
Lane3: T7-R9-GFP-DT
Lane4: T7-R9-FT-DT
Lane5: T7-R9-FT-DT
Lane6: T7-R9-FT-DT
Lane7: T7-R9-FT-DT
Lane8: T7-R9-FT-DT
A member of secretion group electrophoresed DNA from Lane9 to Lane12.
Lane13: T7-His-FT
Lane14: T7-His-FT
Lane15: T7-His-FT
Lane16: T7-His-FT
Lane17: ladder
えいどうしゃしん
Liquid culture(3ml) 3:30~
T7-R9-FT-DT×2, T7-His-FT×2, T7-His-FT
September 16
Miniprep
①T7-R9-FT-DT 150ng/µL
②T7-R9-FT-DT 139ng/μL
③T7-R9-GFP-DT 67ng/µL
④T7-His-FT 153ng/μL
⑤T7-His-FT 73ng/μL
Electrophoresis
September 17
Purifying column
=>FT without RBS :36.4ng/µL
Ligation
Vector(PSB1C3: 12.5/µL, 2000bp) : 3µL = 9fmol
Insert(FT without RBS: 36.4ng/µL, 600bp ): 3µL = 92fmol
Ligation High ver.2 : 3µL
=> 16℃, 1 hours
September 18
Transformation by NAKAGAWA
competent cell | DNA | plate |
---|---|---|
20µL | PSB1C3 FT without RBS 1µL | CP+ |
Verification of R9 function by TAKEUCHI
R9(20µg/µL) | 0.9µL |
GFP(1.2mg/mL) | 2.23µL |
RBS | 16.85µL |
total | 20µL |
X5
Method:
1. Peel cuticles on parafilm by using the head of pencil.(Menasha,wI,54952)
2. Put plant cells into GFP&R9 or GFP for 5~30min.
3. Put plant cells into PBS.
4. Hoechst dyeing.
1 | 2 | 3 | 4 | 5 | 6 | |
R9 | o | o | o | x | o | o |
cuticle | o | o | o | o | x | x |
soak in GFP | 5min | 15min | 30min | 5min | 5min | 30min |
September 19
Transformation(re) by NAKAGAWA,TAKEUCHI
competent cell:20µL
DNA(No RBS FT):1µL
plate :CP+
Transformation(re;re) by NAKAGAWA,TAKEUCHI
competent cell:10µL
DNA(No RBS FT):1µL
LB :100µL
plate :CP+
Adjusted GM Agar Medium making by TAKEUCHI
Ingredient of MS medium(SIGMA M5519): 0.88g
MES : 0.1g
ion exchanged water : 200mL
NaOH : 26µL(adjust to pH5.6)
Agar medium : 1.6g
Autoclave 120℃
Colony PCR(re)
gelA
Lane1: 100bp Ladder
Lane2: No RBS FT colony number 9
Lane3: No RBS FT colony number 10
Lane4: No RBS FT colony number 11
Lane5: No RBS FT colony number 12
Lane6: No RBS FT colony number 13
Lane7: empty
Lane8: empty
gelB
Lane1: 100bp Ladder
Lane2: No RBS FT colony number 14
Lane3: No RBS FT colony number 15
Lane4: No RBS FT colony number 16
Lane5: No RBS FT colony number 17
Lane6: No RBS FT colony number 18
Lane7: No RBS FT colony number 19
Lane8: empty
gelC
Lane1: 100bp Ladder
Lane2: No RBS FT colony number 20
Lane3: No RBS FT colony number 21
Lane4: No RBS FT colony number 22
Lane5: No RBS FT colony number 23
Lane6: No RBS FT colony number 24
Lane7: empty
Lane8: empty
September 20
Liquid culture by NAKAGAWA
No RBS FT x8 (by using September 18)
Plate: CP+
Colony PCR
No RBS FT by using September19
Lane1 : 100bp ladder
Lane2 : colony number 1
Lane3 : colony number 2
Lane4 : colony number 3
Lane5 : colony number 4
Lane6 : colony number 5
Lane7 : colony number 6
Lane8 : colony number 7
Lane9 : colony number 8
Lane10: empty
Lane11: empty
Lane12: 100bp ladder
Liquid culture
A-11, A-12
Miniprep
No RBS FT(by using September20)
1.-10.4µg/mL
2.-4.7µg/mL
3.-3.6µg/mL
4.-7.6µg/mL
5.-4.5µg/mL
6.-8.4µg/mL
7. 1.8µg/mL(average)
8. 18.1µg/mL
Restriction enzyme processing
DNA(No RBS FT)x2 | E.coli | BufferH | MilliQ | total |
---|---|---|---|---|
30µL | 1µL | 4µL | 5µL | 40µL |
in 37℃,2hours
Electrophoresis
DNA(No RBS FT) sample7,8: 10µL
Loading Dye : 2µL
Lane1: 1kb ladder
Lane2: empty
Lane3: No RBS FT(E) sample7
Lane4: empty
Lane5: No RBS FT(E) sample8
Lane6: empty
Electrophoresis(re)
RNA Extraction
5 leaves: 100mg
ISOGEN : 1mL
Elution : 20µL
cDNA Synthesis(1/4)
gDNA wipeout buffer | template RNA | H2O | Total |
---|---|---|---|
1µL | 0.5µL | 5.5µL | 7µL |
Reverse Transcriptase | RT buffer | primer mix | template | Total |
---|---|---|---|---|
0.5µL | 2µL | 0.5µL | 7µL | 10µL |
RT PCR
buffer | dNTPs | MgSO4 | primer f | primer r | Template | KODplus | MilliQ | Total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 2 | 1.5 | 1.5 | 1 | 1 | 34.5 | 50 |
94℃ | 94℃ | 54℃ | 68℃ | cycles |
---|---|---|---|---|
2min | 15sec | 30sec | 10sec | 30 |
1.TUBULIN
2.FUL
3.SEP3
4.AP1
September 22
mRNA extraction
FT and R9
Arabidopsis thaliana's leaves : 200mg
R9 | FT | total |
---|---|---|
1.575μL | 33.425μL | 35μL |
R9 | GFP | |
---|---|---|
1.575 | 33.425 | 35 |
1. Strip cuticles on palafilm by the head of a pencil.
2. Soak plant cells in R9 & FT or R9 & GFP for 2 hours(19:45~).
3. Add PBS (21:45~)
Microscope
R9+GFP+Plant Cell ver.3
R9+
R9 | GFP | PBS | total |
---|---|---|---|
0.9 | 2.25 | 16.85 | 20μL |
R9-
R9 | GFP | PBS | total |
---|---|---|---|
0 | 2.25 | 17.65 | 20 |
same as 9/20, only not on parafilm but on microscope slide from the start.
September 23
RNA Extraction
FT- (GFP+)
FT+
ISOGEN | chloroform | elution | total |
---|---|---|---|
1 | 200 | 11 | 212 |
Reverce Transcription
gDNA wipeout buffer | RNA | total |
1 | 6 | 7 |
at 42 degree, for 2min.
Reverce Transcriptase | RT buffer | primer mix | Template | total |
---|---|---|---|---|
0.5 | 2 | 0.5 | 7 | 10 |
at 42 degree, for 30min.
at 95 degree, for 3min.
RT-PCR
Buffer | dNTPs | MgSO4 | primer | Template | KOD Plus | MilliQ | total |
---|---|---|---|---|---|---|---|
5 | 5 | 2 | 1.5 | 1 | 1 | 34.5 |
94℃, 2min
94℃, 15sec
54℃, 30sec
68℃, 10sec
1.NC TUBULIN
2. FUL
3. SEP3
4. AP1
5.FT TUBULIN
6. FUL
7. SEP3
8. AP1
September 24
ScreeningPCR & Electrophoresis
Lane
1. 100bp ladder
2. T7-His-FT ① 9.2ng/μL
3. T7-His-FT ② 11.7ng/μL
4. T7-His-FT ③ 23.6ng/μL
5. T7-His-FT ④ 21.6ng/μL
6. T7-His-FT ⑤ 30.6ng/μL
7. T7-His-FT ⑥ 51.5ng/μL
8. T7-His-FT ⑦ 47.1ng/μL
9. T7-His-FT(A4) 20.0ng/μL
10. T7-His-FT(B1) 18.8ng/μL
11. final2(by_Secretion_group)
12. blank
FT Introduce
FT | R9 | PBS | total |
---|---|---|---|
50 | 2.88 | 11.12 | 64 |
GFP | R9 | PBS | total |
---|---|---|---|
7.2 | 2.88 | 53.02 | 64 |
We took 1 leaf out of 6 individuals of Arabidopsis thaliana that grow three weeks one by one
Leaves 30mg
1.cut off leaves
2.Inject the juice(by terumo-syringe)(Center tip、tuberculin 1nl)
3.store 20 min, then add PBS 600μL
February 7
Preculture
We started preculture at 12:10.
February 8
Liquid culture
We start culturing with 300mL of LB medium.
time | OD600 |
---|---|
12:00 | start |
14:10 | 0.019 |
14:45 | 0.154 |
15:05 | 0.267 |
15:21 | 0.64 |
Making Competent Cell
We made competent cells.
Transformation
pGEM_TAP
LacP (BBa_R0011)
DT (BBa_B0015)
Making Culture Medium Plates
We made 200mL of ampicillin culture, kanamycin culture, and chloramphenicol culture.
Transformation
GFP(BBa_E0040) in pSB1A2
DT(BBa_B0015) in pSB1AK3
ara(BBa_I0500) in pSB2K3
LacP(BBa_R0011) in pSB1A2
February 9
Transformation
BBa-E0040(GFP)(Mr.Fujita)
Liquid culture
DT.leap colony transformed on February 8
colony of competent cell made on February 8
200pxB0040 1.4k PsB1A2 B0034 1.2M pSB1A2(from iGEM parts plate)
Making Competent cells
We did preculture for overnight. We put 1.5mL of preculture on 150mL of LB culture.
time | OD600 |
---|---|
11:45 | start |
13:30 | 0.048 |
14:30 | 0.168 |
15:03 | 0.256 |
15:20 | 0.405 |
15:35 | 0.459 |
at last | 0.576 |
February 11
Checking Transformation efficiency
Conpetent cell's transformation efficiency is 1.3x10^4colonys/μg
February 13
Transformation
Const promoter J23110, J23109, J23100
DNA | Competent cell | total |
---|---|---|
1μL | 20 | 21 |
No colony was there on February 14
Liquid culture
LacP, DT, RBS(BBa_B0034),GFP
start at 20:00
in Plus grow with Ampicilin 3mL
February 14
Miniprep
concentration[μg/μL]
LacP3 | 39.6 |
LacP4 | 40.8 |
LacP5 | 28.9 |
RBS1 | 28.2 |
RBS2 | 57.4 |
RBS3 | 13.2 |
DT3 | 69.7 |
DT4 | 64.4 |
DT5 | 61.5 |
GFP1 | 64.0 |
GFP2 | 50.5 |
GFP3 | 66.0 |
Restrictive Digestion
Const promoterJ23100
DNA | Spe1 | Pst1 | Buffer2 | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
at 37℃ for overnight
February 15
Making gel
1% Agarose gel
Agarose | TAE |
---|---|
1.6g | 160mL |
Electrophoresis
Gel No.1
Restriction product | loading dye |
---|---|
5μL | 1 |
The marker was 1kb ladder
It seemed that this restriction product was not cut.
Gel No.2
Lane1 : 1kb ladder
Lane2 : J23100 2μL + 6*Loading dye 1μL
Lane3 : J23100(Spe1,Pst1) 5μL
Lane4 : J23100(Spe1,Pst1) 2μL
- There were bands on lane_2 and we cannot identify these bands because the sample of lane_2 was not cut with any restriction enzyme.
- There must have been bands at 2100bp and 883bp on lane_3 and lane_4.
Testing whether restriction enzyme were deactivated or not
DNA(DT) | restriction enzyme | Buffer | BSA | MilliQ | total |
---|---|---|---|---|---|
10 | 0.5 | 3 | 0.5 | 16 | 30 |
at 37℃ for Oveernight
Restriction enzyme means Spe1(1,2) Pst1(1,2,3) in this time.
February 16
Electrophoresis
1. 1kb ladder
2. DT2
3. DT3
4. DT2 (Spe1-1)
5. DT2 (Spe1-2)
6. DT2 (Pst1-1)
7. DT2 (Pst1-2)
8. DT2 (Pst1-3)
9. DT3 (Pst1-4)
10. 1kb ladder
Pst1-1, Pst1-2, and Pst1-3 did not cut DNAs. They seemed to be deactivated.
Genomic PCR
10*Buffer for KOD Plus | 2mM dNTPs | 25mM MgSO4 | 10μM primer-f | 10μM primer-r | 158ng/μL Genomic DNA | KOD plus | MilliQ | total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 1 | 32 | 50 |
Electrophoresis
1. 1kb ladder
2. tatABCD (2.5kb)
3. TAMO reductase (2.7kb)
4. Negative control
We got bands of tatABCD but there were nonspecific amplification products.
We failed amplification of TAMO reductase.
Transformation
Constitutive promoter (BBa_J23107 , BBA_J23117)
High copy plasmid (pSB1AT3)
DNA | competent cell |
---|---|
1μL | 10μL |
February 17
PCR
We did PCR to amplify products of PCR that we had done yesterday but we could not amplify tatABCD.
Genomic PCR
Buffer | dNTPs | MgSO4 | primer-f | primer-r | genomic DNA | KOD plus | MilliQ | total |
---|---|---|---|---|---|---|---|---|
2.5 | 2.5 | 5 | 0.75 | 0.75 | 0.5 | 0.5 | 16 | 50 |
Predenature 94℃, 2min
Denature 98℃, 10sec
Annealing 57℃, 30sec
Extension 68℃, 2.5min
(30cycles)
Electrophoresis
1. 1kb ladder
2. TAMO reductase
3. Negative control
Restrictive Digestion
J23100 | Spe1 | Pst1 | Buffer2 | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
Genomic PCR
Buffer | dNTPs | MgSO4 | primer-f | primer-r | genomic DNA | KOD plus | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
TMAO reductase | 2.5 | 2.5 | 3 | 0.75 | 0.75 | 0.5 | 0.5 | 15.5 | 25 |
tatABCD | 2.5 | 2.5 | 2 | 0.75 | 0.75 | 0.5 | 0.5 | 15.5 | 25 |
tatABCD | 2.5 | 2.5 | 2 | 0.75 | 0.75 | 5 | 0.5 | 10.5 | 25 |
Electrophoresis
1. 1kb ladder
2.3. TAMO reductase
4. tatABCD
5. tatABCD(10 times amount of genome)
Checking of restriction enzyme
DT | enzyme | Buffer | BSA | MilliQ | total |
---|---|---|---|---|---|
2 | 0.5 | 3 | 0.5 | 24 | 30 |
at 37℃ for overnight
We checked EcoR1 and Xba1.
February 18
Miniprep
μg/mL | 260/280 | 230/260 | |
---|---|---|---|
JS3117-1 | 135 | 1.5 | 2.06 |
JS3117-2 | 75 | 1.6 | 1.63 |
JS3109-1 | 115 | 1.5 | 1.88 |
JS3109-2 | 75 | 1.65 | 1.71 |
pSB1AT3-1 | 70 | 1.66 | 1.83 |
pSB1AT3-2 | 100 | 1.52 | 1.54 |
diluted to 25 times
Making Competent cells
We put 3mL of preculture product on yesterday onto 300mL of LB medium
time | OD600 |
---|---|
10:30 | start |
12:10 | 0.118 |
13:00 | 0.270 |
13:30 | 0.502 |
Transformation
pSB1AT3-2 | competent cell | MilliQ | total |
---|---|---|---|
0 | 20 | 10 | 30 |
2 | 20 | 8 | 30 |
10 | 20 | 0 | 30 |
- Results(on Feb. 19)
pSB1AT3-2 | number of colony |
---|---|
0 | 0 |
2 | 177 |
10 | 590 |
Transformation efficiency 7.4x10^4 colonys/μg
February 20
Restrictive Digestion
sample 1
DT plasmid | EcoR1 | Xba1 | Buffer | BSA | MilliQ | total |
---|---|---|---|---|---|---|
7.5 | 0.5 | 0.5 | 3 | 0.5 | 18 | 30 |
sample2
GFP plasmid | EcoR1 | Spe1 | Buffer | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
Electrophoresis
- sample1
a. sample1 2μL + MilliQ 3μL + 6×Loading Dye 1μL
b. sample1 5μL + 6×Loading Dye 1μL
lane1. 1kb ladder
lane2. a
lane3. b
lane4. a
lane5. b
lane6. a
lane7. b
lane8. 1kb ladder
- sample2
c. sample2 2μL + MilliQ 3μL + 6×Loading Dye 1μL
d. sample2 5μL + 6×Loading Dye 1μL
lane1. 1kb ladder
lane2. c
lane3. d
lane4. 1kb ladder
PCR
Buffer | dNTPs | MgSO4 | primer-f | primer-r | genomic DNA | KOD plus | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
tatABCD1 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 0.5 | 16 | 25 |
tatABCD2 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 0.5 | 16 | 25 |
TMAO reductase1 | 2.5 | 2.5 | 2 | 0.75 | 0.75 | 0.5 | 0.5 | 15.5 | 25 |
TMAO reductase2 | 2.5 | 2.5 | 2 | 0.75 | 0.75 | 5 | 0.5 | 10.5 | 25 |
Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 2.5min
→30cycles
Buffer | dNTPs | MgSO4 | primer-f | primer-r | PCR products | genomic DNA | KOD plus | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|---|
tatABCD1 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0 | 0.5 | 0.5 | 16 | 25 |
tatABCD2 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 1 | 0 | 0.5 | 15.5 | 25 |
TMAO reductase1 | 2.5 | 2.5 | 2 | 0.75 | 0.75 | 0 | 0 | 0.5 | 16 | 25 |
TMAO reductase2 | 2.5 | 2.5 | 2 | 0.75 | 0.75 | 0 | 0 | 0.5 | 16 | 25 |
Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 2.5min
→35cycles
Electrophoresis
lane1.1kb ladder
lane2.tatABCD
lane3.tatABCD
lane4.TAMO1
lane5.TAMO2
lane6.1kb ladder
lane1.1kb ladder
lane2.tatABCD1
lane3.tatABCD2
lane4.TAMO
lane5.TAMO
lane6.1kb ladder
February 21
PCR (Advantage HF protocol)
buffer | dNTPs | primer-f | primer-r | gDNA | PCR products | DW | polymerase | total | |
---|---|---|---|---|---|---|---|---|---|
tatABCD | 2.5 | 2.5 | 0.75 | 0.75 | 1 | 0 | 17 | 0.5 | 25 |
TMAO | 2.5 | 2.5 | 0.75 | 0.75 | 0 | 1 | 17 | 0.5 | 25 |
Predenature 94℃ 1min
Denature 94℃ 30sec
Annealing 58℃ 30sec
Extension 68℃ 3min
→25cycles
Electrophoresis
1. 1kb ladder 2μL
2. tatABCD 5μL + 6×Loading Buffer 1μL
3. TAMO 5μL + 6×Loading Buffer 1μL
4. 1kb ladder 2μL
Liquid culture
pSB3C5-1,2
pSB4K5-1,2
February 22
Gel extraction
lane 1 of the gel 45.0μg/mL
PCR purification
product 38.2μg/mL
PCR
TMAO reductase
Buffer | dNTPs | MgSO4 | prefix primer-f | suffix primer-r | product of gel extract | product of PCR purification(1ng/μL) | KOD plus | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|---|
1 | 5 | 5 | 4 | 1.5 | 1.5 | 0.5 | 0 | 1 | 32.5 | 50 |
2 | 5 | 5 | 4 | 1.5 | 1.5 | 1 | 0 | 1 | 32.5 | 50 |
3 | 5 | 5 | 4 | 1.5 | 1.5 | 0 | 0.5 | 1 | 32.5 | 50 |
4 | 5 | 5 | 4 | 1.5 | 1.5 | 0 | 1 | 1 | 32.5 | 50 |
94℃, 2min
98℃, 10sec
59℃, 30sec
68℃, 3min
→25cycles
Electrophoresis
1. 1kb ladder
2. TAMO1
3. TAMO2
4. TAMO3
5. TAMO4
6. constructive promoter 1-18C
7. constructive promoter Spe1
8. constructive promoter Pst1
9. 1kb ladder
PCR
Buffer | dNTPs | MgSO4 | prefix primer-f | suffix primer-r | product of PCR purification(1ng/μL) | KOD plus | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
1 | 5 | 5 | 3 | 1.5 | 1.5 | 0.5 | 1 | 32.5 | 50 |
2 | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 1 | 32 | 50 |
3 | 5 | 5 | 3 | 1.5 | 1.5 | 2 | 1 | 31 | 50 |
4 | 5 | 5 | 3 | 1.5 | 1.5 | 3 | 1 | 30 | 50 |
5 | 5 | 5 | 3 | 1.5 | 1.5 | 10 | 1 | 29 | 50 |
6 | 5 | 5 | 3 | 1.5 | 1.5 | 0 | 1 | 33 | 50 |
94℃, 2min
98℃, 10sec
59℃, 30sec
68℃, 3min
→25cycles
Electrophoresis
Checking Dpn1
Buffer | LacP(28.7ng/μL) | Dpn1 | MilliQ | total |
---|---|---|---|---|
2 | 10 | 0.5 | 7.5 | 20 |
2 | 10 | 0 | 5 | 17 |
February 23
Colony PCR
tatABCD(2 samples)
Buffer | dNTPs | MgSO4 | primer-f | primer-r | KOD plus | MilliQ | total |
---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |
Predenature 94℃ 1min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 3min
→25cycles
Electrophoresis
1. 1kb ladder 2μL
2. tatABCD 1 5μL + 6×Loading Buffer 1μL
3. tatABCD 2 5μL + 6×Loading Buffer 1μL
4. 1kb ladder 2μL
Miniprep
pSB4K5 and pSB3C5
deluted it to 25 times and then measured it
pSB4K5 1 : 60.0 μg/ml 1.67(260/280) 1.98(260/230)
pSB4K5 2 : 55.0 μg/ml 1.49(260/280) 1.62(260/230)
pSB3C5-3 : 3.6 μg/ml 1.57(260/280) 3.00(260/230)
pSB3C5-4 : 1.3 μg/ml 1.44(260/280) 1.04(260/230)
Liquid culture
pSB3C5-3,4
Electrophoresis
1. 1kb ladder 2μL
2. pSB3C5-3 5μL, 6×loading dye 1μL
3. pSB3C5-4 5μL, 6×loading dye 1μL
4. 1kb ladder 2μL
February 27
Test of Dpn1
Buffer2 | GFP2 | BSA | MilliQ | Dpn1 |
---|---|---|---|---|
3 | 3 | 0.3 | 23 | 1 |
Colony PCR
buffer | dNTPs | MgSO4 | Primer-f | Primer-r | MilliQ | KOD Plus | total | |
---|---|---|---|---|---|---|---|---|
Colony PCR(2 samples) | 5 | 5 | 3 | 1.5 | 1.5 | 33 | 1 | 50 |
Negative control | 5 | 5 | 3 | 1.5 | 1.5 | 34 | 0 | 50 |
Predenature 94℃,2min
Denature 98℃,10sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles
Electrophoresis
lane | DNA | sample | Loading Dye | MilliQ |
---|---|---|---|---|
1 | 1kb ladder | 2 | 0 | 0 |
2 | product of PCR1 | 5 | 1 | 0 |
3 | product of PCR2 | 5 | 1 | 0 |
4 | product of PCR(Negative control) | 5 | 1 | 0 |
5 | product of PCR(2/23) | 5 | 1 | 0 |
6 | GFP2(DPN1) | 10 | 2 | 0 |
7 | GFP2 | 3 | 2 | 7 |
8 | 1kb ladder | 2 | 0 | 0 |
Results of liquid culture
We measure this after dilute it to 10 times.
pSB3C5-5 | pSB3C5-6 | pSB3C5-5(1% glucose) | pSB3C5-6(1% glucose) |
---|---|---|---|
8.5[µg/ml] | -1.8 | -17.9 | -18.2 |
PCR
buffer | dNTPs | MgSO4 | Primer-f(prefix) | Primer-r(suffix) | PCR purification product(1ng/µL) | MilliQ | KOD Plus | total | |
---|---|---|---|---|---|---|---|---|---|
1 | 5 | 5 | 3 | 1.5 | 1.5 | 0.2 | 32.8 | 1 | 50 |
2 | 5 | 5 | 3 | 1.5 | 1.5 | 0.5 | 32.5 | 1 | 50 |
- PCR purification product was that purification product(75ng/µL) of electrophoresis-3 deluted to 1ng/µL
Predenature 94℃,2min
Denature 98℃,10sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles
February 28
Electrophoresis
1. 1kb ladder
2. PCR1 →Product of gel extraction : tatABCD with prefix and suffix 105[ng/µL]
3. PCR2
Restrictive Digestion
Buffer2 | plasmid(?) | enzyme | MilliQ | total |
---|---|---|---|---|
2 | 2 | 0.2 | 15.8 | 20 |
incubate 1 hour at 37℃
Electrophoresis
1. 1kb ladder
2. Control (without enzymes)
3. EcoR1
4. Xba1 (crystallized)
5. Xba1 (with seal)
6. Spe1
7. Pst1
8. 1kb ladder
PCR and Electrophoresis
Quick Taq Dye Mix | primer-f | primer-r | template | MilliQ | total |
---|---|---|---|---|---|
25 | 1.0 | 1.0 | 0.5 | 22.5 | 50 |
Predenature 94℃,2min
Denature 94℃,30sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles
Restrictive Digestion
BufferH | tatABCD | EcoR1 | Spe1 | MilliQ | total |
---|---|---|---|---|---|
2 | 5 | 0.2 | 0.2 | 12.6 | 20 |
PCR purification
We eluted the product for 30µL MilliQ
Ligation
Insert(tatABCD) | Vector(pSB1C3) | Ligation High | total |
---|---|---|---|
10 | 1 | 5 | 16 |
4℃, overnight
February 29
Transformation
tatABCD | competent cell | total |
---|---|---|
1 | 10 | 11 |
Checking Restriction enzyme
plasmid seems to be 1-18C promoter | Enzyme | Buffer | MilliQ | total |
---|---|---|---|---|
2 | 0.2 | 2 | 15.8 | 20 |
Checking tatABCD
tatABCD | Hind3 | Buffer | MilliQ | total |
---|---|---|---|---|
5 | 0.2 | 2 | 12.8 | 20 |
PCR
buffer | dNTPs | MgSO4 | Primer-f | Primer-r | ColE1(6.5ng/µL) / TMAO | MilliQ | KOD Plus Neo | total | |
---|---|---|---|---|---|---|---|---|---|
Kil | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 16 | 0.5 | 25 |
TMAO | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 16 | 0.5 | 25 |
Predenature 94℃,2min
Denature 98℃,10sec
Annealing 60℃,30sec
Extension 68℃,3min
→30cycles
Electrophoresis
1. 1kb ladder
2. Kil (649bp)
3. TMAO (2720bp)
4. TMAO (Quick Taq)
5. tatABCD (Quick Taq)
6. tatABCD (Hind3)
7. 1kb ladder
March 1
PCR
- TMAO
Template is gDNA and product of colony PCR gel extraction
Buffer | gNTPs | MgSO4 | Primer-f | Primer-r | KOD Plus Neo | Template gDNA | product of gel extraction | DW | total | |
---|---|---|---|---|---|---|---|---|---|---|
1 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 0.5 | 0 | 16 | 25 |
2 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 0 | 2 | 14.5 | 25 |
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 1.5min
→25cycles
- Kil
Buffer | dNTPs | MgSO4 | primer-f | primer-r | colE1(6.5ng/µL) | KOD Plus Neo | MilliQ | total |
---|---|---|---|---|---|---|---|---|
2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 16 | 0.5 | 25 |
94℃, 2min
98℃, 10sec
61℃, 30sec
68℃, 1min
→20cycles
Electrophoresis
1. 1kb ladder
2. TMAO1 (gDNA)
3. TMAO2 (product of gel extraction)
4. Kil
PCR
- kil
Buffer | dNTPs | MgSO4 | primer-f | primer-r | Product of Purification | KOD Plus Neo | MilliQ | total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50 |
94℃, 2min
98℃, 10sec
61℃, 30sec
68℃, 30sec
20cycles
→Purification 230ng/µL
Restrictive Digestion
Kil | EcoR1 | Spe1 | BufferH | MilliQ | total |
---|---|---|---|---|---|
5 | 0.2 | 0.2 | 2 | 12.6 | 20 |
incubate at 37℃, for 1.5 hours
PCR Purification
Ligation
Kil | pSB1C3 | Ligation High | total |
---|---|---|---|
5 | 1 | 3 | 9 |
at 4℃, for overnight
March 2
Ligation
Kil | pSB1C3 | Ligation High Ver.2 | total |
---|---|---|---|
5 | 1 | 3 | 9 |
tatABCD | pSB1C3 | Ligation | total |
---|---|---|---|
10 | 1 | 5 | 16 |
at 16℃ for 1 hour
Transformation
Kil | Kil(3/1,Ligation) | tatABCD | competet cell | total |
---|---|---|---|---|
1 | 0 | 0 | 10 | 11 |
0 | 1 | 0 | 10 | 11 |
0 | 0 | 1 | 10 | 11 |
PCR
Quick Taq | primer-r | primer-f | template | MilliQ | total |
---|---|---|---|---|---|
25 | 1 | 1 | 0.5 | 22.5 | 50 |
Electrophoresis
Restrictive Digestion
pSB3C5-5 | EcoR1 | Pst1 | BufferH | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
at 37℃, for 2 hour
Electrophoresis
1. 1kb ladder 2µL
2. pSB3C5 5µL + 6×Loading Buffer 1µL
・product of gel extraction(about 2700bp)
-30.9µg/mL
Restrictive Digestion
GFP1 ,2 ,3
GFP | EcoR1 | Pst1 | Buffer | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
at 37℃, for 2.5 hours
Restrictive Digestion
DT | EcoR1 | Xba1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.2 | 0.2 | 3 | 0.3 | 16.3 | 30 |
Constitutive Promoter | Spe1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
15 | 0.2 | 0.2 | 3 | 0.3 | 11.3 | 30 |
at 37℃, 2 hours
- J23117-1:135ng/µL, J23107-1:115ng/µL
- DT3→PCR Purification
- Promoter→Gel Extraction
Checking TMAO
something seems to be TMAO | Buffer2 | EcoR1 | MilliQ | total |
---|---|---|---|---|
10 | 2 | 0.5 | 7.5 | 20 |
at 37℃, for 1 hour
Electrophoresis
1. 1kb ladder
2. GFP1 that had been cut by restriction enzyme
3. GFP2 that had been cut by restriction enzyme
4. GFP3 that had been cut by restriction enzyme
5. GFP1
6. GFP2
7. GFP3
8. TMAO (control)
9. TMAO (EcoR1)
10. DT (control)
11. DT (EcoR1, Xba1)
12. 1kb ladder
Checking tatABCD
Quick Taq | primer-f | primer-r | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
Electrophoresis
March 3
PCR
template | buffer | dNTPs | MgSO4 | VF | VR | KOD plus | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
1 | 1 | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 50 |
2 | 2 | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 31 | 50 |
94℃, 2min
98℃, 10sec
50℃, 30sec
68℃, 1min
→30cycles
Miniprep
14.2µg/mL, 15.0µg/mL
March 4
Sequence of tatABCD
Quick Taq | primer-f | promer-r sequence | template | MilliQ | total |
---|---|---|---|---|---|
25 | 1 | 1 | 1 | 23 | 50 |
Quick Taq | primer-f sequence | primer-r | template | MilliQ | total |
---|---|---|---|---|---|
25 | 1 | 1 | 1 | 23 | 50 |
Colony PCR of TMAO
buffer | dNTPs | NgSO4 | primer-f | primerr-r | KOD plus | MilliQ | total |
---|---|---|---|---|---|---|---|
5 | 5 | 4 | 1.5 | 1.5 | 1 | 32 | 50 |
→ethanol precipitation
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 2.5min
→25cycles
Electrophoresis
1. 1kb ladder
2. tatABCD1
3. tatABCD2
4. TMAO
5. 1kb ladder
Restrictive Digestion
TMAO | EcoR1 | BufferH | BSA | MilliQ | total |
---|---|---|---|---|---|
10 | 0.2 | 3 | 0.3 | 16.5 | 30 |
TMAO | Xba1 | Pst1 | BudderM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.2 | 0.2 | 3 | 0.3 | 16.3 | 30 |
at 37℃ for 1 hour
Electrophoresis
Transformation
pSB1C3 | competent cell(made at 2/8) | total |
---|---|---|
5 | 100 | 105 |
March 5
Restrictive Digestion
pSB1C3(Xba1, Spe1) | Pst1 | BufferH | BSA | MilliQ | total |
---|---|---|---|---|---|
10 | 0.2 | 2 | 0.2 | 7.6 | 20 |
pSB1C3(Xba1, Spe1) | EcoR1 | BufferH | BSA | MilliQ | total |
10 | 0.2 | 2 | 0.2 | 7.6 | 20 |
at 37℃ for 1 hour
→Then we did ethanol precipitation
Ligation
Kil(EcoR1, Spe1) | pSB1C3(EcoR1) | Ligation High | total |
---|---|---|---|
5 | 1 | 3 | 9 |
at 16℃ for 1 hour
Transformation
Kil | competent cell | total |
---|---|---|
1 | 10 | 11 |
We used commercially available competent cells in this time.
PCR
TMAO
buffer | dNTPs | MgSO4 | Primer-f | Primer-r | Template | KOD plus | MilliQ | total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 1 | 32 | 50 |
Electrophoresis
Restrictive Digestion
LacP | pSB3C5 | EcoR1 | Pst1 | BufferH | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|
1 | 20 | 0 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
2 | 0 | 20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
at 37℃ for 1 hour
1→Ethanol precipitation 45.4µg/mL
2→Gel extraction 38.7µg/mL
Ligation
LacP | pSB3C5 | Ligation High | total |
---|---|---|---|
10 | 2 | 6 | 18 |
at 4℃ for overnight
Transformation
LacP+pSB3C5 | competent cell | total |
---|---|---|
1 | 10 | 11 |
on ice for 30 mins.
heat shock at 42℃ for 60secs
on ice for 2 mins.
After we incubate with 200µL of SOC culture for 1 hour, we did plating on LB culture with CP
Restrictive Digestion
GFP Plasmid | EcoR1 | Spe1 | Buffer2 | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
at 37℃ for 2 hours
Electrophoresis
1. Ladder 2µL
2. GFP Plasmid (already restricted) 2µL + Loading Dye 2µL + MilliQ 8µL
3. Ladder 2µL
PCR
torA signal and pspA
We did Colony PCR to pspA
Buffer | dNTPs | MgSO4 | Primer-f | Primer-r | template(TMAO) | KOD plus | MilliQ | total |
---|---|---|---|---|---|---|---|---|
2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 0.5 | 16 | 25 |
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
electrophoresis
1. 100bp Ladder
2. torA signal (272bp)
3. pspA (969bp)
4. 100bp Ladder
March 6
Restrictive Digestion
GFP | EcoR1 | Spe1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
12 | 0.5 | 0.5 | 3 | 0.5 | 13.5 | 30 |
We did incubate at 37℃ for 1.5hours.
And we did gel extraction on 3/7 and get 40.0μg/mL GFP.
PCR
buffer | dNTPs | MgSO4 | Primer-f | Primer-r | template | KOD plus neo | MilliQ | total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 0.5 | 1 | 32.5 | 50 |
94℃ 2min
98℃ 10sec
60℃ 30sec
68℃ (torA 10sec / pspA 30sec) 25 cycles
We used product of PCR on 3/5 of torA and pspA as template.
Restrictive Digestion
Kil | EcoR1 | Spe1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.3 | 0.3 | 3 | 0.3 | 16.1 | 30 |
DT | EcoR1 | Xba1 | Buffer2 | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
at 37℃ for 2 hours
→ purification 37.7ng/μL
Ligation
Kil | pSB1C3 | Ligation High Ver.2 | total |
---|---|---|---|
4 | 2 | 3 | 9 |
- Kil : 350fmol
- pSB1C3 : 29fmol
at 16℃ for overnight
March 7
Electrophoresis
1. 1kb ladder 2µL
2. pspA (PCR product)2.5µL + Loading Dye 0.5µL
3. GFP 30µL + Loading Dye 6µL
4. 1kb ladder 2µL
- The GFP was gel extracted on 3/6 and concentrated by Vacuum in 150µg/mL
Ligation
VectorDNA | GFP | Ligation High Ver.2 | total |
---|---|---|---|
5 | 15 | 10 | 30 |
Restrictive Digestion
torA | EcoR1 | Spe1 | bufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.3 | 0.3 | 3 | 0.3 | 16.1 | 30 |
pspA | EcoR1 | Spe1 | bufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
5 | 0.3 | 0.3 | 3 | 0.3 | 21.1 | 30 |
at 37℃ for 1.5 hours
Purification
torA→31.8ng/µL
pspA→49.3ng/µL
Ligation
torA | pSB1C3 | Ligation High Ver.2 | total |
---|---|---|---|
3 | 3 | 3 | 9 |
pspA | pSB1C3 | Ligation High Ver.2 | total |
---|---|---|---|
4 | 2 | 3 | 9 |
at 4℃, for overnight
- torA→31.8ng/µL×3µL=95.4ng=0.529pmol
- pSB1C3→19.4ng/µL×3µL=58.2ng=0.042pmol
- pspA→49.3ng/µL×4µL=197.2ng=0.308pmol
- pSB1C3→19.4ng/µL×2µL=38.8ng=0.029pmol
March 8
Restrictive Digestion
pSB4K5 | EcoR1 | Spe1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.2 | 0.2 | 3 | 0.2 | 6.4 | 30 |
at 37℃ for 1 hour.
→Purification : 36.6ng/µL
Ligation
Kil | pSB4K5 | Ligation High Ver.2 | total |
---|---|---|---|
10 | 1 | 5 | 16 |
at 4℃ for overnight
- Kil→37.7ng/µL×10µL=377ng=879fmol
- pSB4K5→36.6ng/µL×1µL=36.6ng=86fmol
Liquid culture
LacP + pSB3C5 -1, 2
Transformation
torA | pspA | competent cell | total |
---|---|---|---|
1 | 0 | 10 | 11 |
0 | 1 | 10 | 11 |
We use commercially available competent cells in this time.
March 9
Restrictive Digestion
tatABCD | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.2 | 0.2 | 3 | 0.3 | 16.3 | 30 |
at 37℃ for 1hour
→purification : 75.0μg/mL (1.11 260/280 , 0.81 260/230)
Miniprep
LacP + pSB3C5 1 80.5μg/mL (1.78 260/280 , 2.00 260/230)
LacP + pSB3C5 2 107.2μg/mL (1.83 260/280 , 1.90 260/230)
Colony PCR
Quick Taq | VF | VR | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃ 2min
94℃ 30sec
55℃ 30sec
68℃ 6sec
25cycles
Ligation
tatABCD | constP J23107 | Ligation High Ver.2 | total |
---|---|---|---|
5 | 1 | 3 | 9 |
tatABCD : 227fmol
constP J23107 : 21fmol
Transformation
pspA | torA | Kil | competent cell | |
---|---|---|---|---|
1 | 1 | 0 | 0 | 10 |
2 | 0 | 1 | 0 | 10 |
3 | 0 | 0 | 1 | 10 |
Miniprep
4mL of plusgrow which had been cultured for overnight.
pSB4K5 : 80.5μg/mL
March 10
Screening PCR
Quick Taq | VF | VR | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
Then we did electrophoresis to confirm.
1.1kb ladder
2.kil (649bp)
3,4,5, pspA (969bp)
6,1kb ladder
1, 100bp ladder
2,3,4, torA signal
Restrictive Digestion
LacP-pSB3C5 | Spe1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.2 | 0.2 | 2 | 0.2 | 7.4 | 20 |
for 2.5 hours at 37℃
→purification 29.0ng/μL
torA | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.2 | 0.2 | 2 | 0.2 | 7.4 | 20 |
for 2.5 hours at 37℃
→purification 91.8ng/μL
Ligation
torA | LacP-pSB3C5 | Ligation High Ver.2 | total |
---|---|---|---|
4 | 2 | 3 | 9 |
torA : 929fmol
LacP-pSB3C5 : 284fmol
for overnight at 4℃
March 11
Miniprep
We used 3μL of plus grow that we had cultured for overnight.
torA : 62.8ng/μL
Screening PCR
Quick Taq | VF | VR | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
Electrophoresis
1. 1kb ladder
2〜11. pspA (969bp)
12. 1kb ladder
The results were shown as photograph in the right.
It seemed that there were shorter sample than expected sample, so we did electrophoresis with pspA which was product of PCR and pspA which had already cut with EcoR1 and Spe1.
1.1kb ladder
2. pspA (PCR product)
3, pspA (Eco, Spe)
4〜6, pspA (colony PCR)
7,1kb ladder
The results were shown as photograph in the right.
March 12
Transformation
DT(1ng/μL) | DT(0.1ng/μL) | Kil | LacP-torA | MilliQ | competent cell | total |
---|---|---|---|---|---|---|
1 | 0 | 0 | 0 | 0 | 20 | 21 |
0 | 1 | 0 | 0 | 0 | 20 | 21 |
0 | 0 | 5 | 0 | 0 | 50 | 51 |
0 | 0 | 0 | 5 | 0 | 50 | 51 |
0 | 0 | 0 | 0 | 1 | 20 | 21 |
Restrictive Digestion
pspA | EcoR1 | Spe1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.3 | 15.7 | 30 |
at 37℃ for 4 hours
→ We did purification and got 48.3ng/μL pspA.
Ligation
pspA | pSB1C3 | MilliQ | Ligation High Ver.2 | total |
---|---|---|---|---|
4 | 2 | 0 | 3 | 9 |
2 | 2 | 0 | 2 | 6 |
0 | 2 | 2 | 2 | 6 |
March 13
Miniprep
pSB1C3 74.2µg/mL 1.65 (260/280) 1.31 (260/230)
March 14
Restrictive Digestion
pSB1C3 | EcoR1 | Spe1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.2 | 0.2 | 4 | 0.4 | 15.2 | 40 |
We did gel extraction and got 47.2ng/µL pSB1C3 but we did not cut out RFP.
Ligation
torA | pSB1C3 | Ligation High Ver.2 | total |
---|---|---|---|
4 | 2 | 3 | 9 |
MilliQ | pSB1C3 | Ligation High Ver.2 | total |
4 | 2 | 3 | 9 |
at 16℃, for 1 hour
- torA : 0.707pmol
- pSB1C3 : 0.068pmol
Liquid culture
We cultured LacP-pSB3C5 1,2,3,4,5 (CP tolerance)on culture with Amp.
→Only 4 which did not be cultured succeeded.
March 15
Liquid culture
We cultured LacP-pSB3C5 6,7,8,9,10 on culture with ampicillin.
→6,8,9,10 were succeeded.
Restrictive Digestion
GFP | EcoR1 | Spe1 | Buffer2 | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
DT | EcoR1 | Xba1 | Buffer2 | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
for 2 hours at 37℃.
Miniprep
pspA (pSB1C3) 40.5ng/µL
Ligation
pspA | DT | Ligation High Ver.2 | total |
---|---|---|---|
5 | 1.5 | 3 | 9.5 |
- pspA : 385fmol
- DT : 36fmol
Transformation
J23107-tatABCD | DT (0.1ng/µL) | DT (0.01ng/µL) | pspA-DT | competent cells on 3/15 | total |
---|---|---|---|---|---|
2 | 0 | 0 | 0 | 20 | 22 |
0 | 2 | 0 | 0 | 20 | 22 |
0 | 0 | 2 | 0 | 20 | 22 |
0 | 0 | 0 | 2 | 20 | 22 |
Screening PCR
Kil, pspA and torA
Quick Taq | Primer-r | Primer-f | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
March 16
Miniprep
torA (pSB1C3) 68.8ng/µL
Kil (pSB4K5) 92.7ng/µL
torA was red for some reason. We do not know why.
Colony PCR
Quick Taq | Primer-r | Primer-f | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 6sec
→25cycles
Electrophoresis
The results were shown as photograph in the right.
Checking Transformation Efficiency
competent cells that were made on March 15.
DNA : 0.02ng → 668 colonies Transformation Efficiency : 3.3×10^7
DNA : 0.2ng → 1739 colonies Transformation Efficiency : 8.7×10^6
Restrictive Digestion
pSB1C3 | EcoR1 | Spe1 | BSA | BufferM | BufferH | MilliQ | total |
---|---|---|---|---|---|---|---|
20 | 0.2 | 0.2 | 0.3 | 3 | 0 | 6.3 | 30 |
5 | 0.2 | 0 | 0.2 | 0 | 2 | 12.6 | 20 |
at 37℃, for 2 hours.
We did gel extraction for product with EcoR1, Spe1. We got 46.1ng/µL pSB1C3.
Kil(pSB4K5) | EcoR1 | Pst1 | BSA | BufferH | MilliQ | total |
---|---|---|---|---|---|---|
5 | 0.2 | 0.2 | 0.2 | 2 | 12.4 | 20 |
5 | 0.2 | 0 | 0.2 | 2 | 12.6 | 20 |
for overnight at 37℃.
We did this to confirm.
pspA (pSB1C3) | EcoR1 | Pst1 | BSA | BufferH | MilliQ | total |
---|---|---|---|---|---|---|
5 | 0.2 | 0.2 | 0.2 | 2 | 12.4 | 20 |
5 | 0.2 | 0 | 0.2 | 2 | 12.6 | 20 |
for overnight at 37℃.
We did this to confirm.
Ligation
torA | pSB1C3 | Ligation High Ver.2 | total |
---|---|---|---|
4 | 2 | 3 | 9 |
MilliQ | pSB1C3 | Ligation High Ver.2 | total |
---|---|---|---|
4 | 2 | 3 | 9 |
at 16℃, for overnight
- torA→767fmol
- pSB1C3→68fmol
March 17
Miniprep
J23107-tatABCD 72.7ng/µL
pspA-DT 50.5ng/µL
Checking the Insert
J21037-tatABCD | EcoR1 | Pst1 | BSA | BufferH | MilliQ | total |
---|---|---|---|---|---|---|
5 | 0.2 | 0.2 | 0.2 | 2 | 12.4 | 20 |
5 | 0.2 | 0 | 0.2 | 2 | 12.6 | 20 |
Success.
pspA-DT | EcoR1 | Pst1 | BSA | BufferH | MilliQ | total |
---|---|---|---|---|---|---|
5 | 0.2 | 0.2 | 0.2 | 2 | 12.4 | 20 |
5 | 0.2 | 0 | 0.2 | 2 | 12.6 | 20 |
Failed.
March 19
Restrictive Digestion
DT | EcoR1 | Xba1 | BSA | BufferM | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.2 | 0.2 | 0.3 | 3 | 16.3 | 30 |
We did Gel extraction and got 17.2ng/µL of DT.
Ligation
Kil | DT | Ligation High Ver.2 | total |
---|---|---|---|
10 | 2 | 6 | 18 |
We did this for an hour at 16℃.
Restrictive Digestion
GFP | EcoR1 | Spe1 | BSA | BufferM | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 0.5 | 3 | 15.5 | 30 |
We did this for 4 hours at 37℃
Ligation
pspA | DT | Ligation High Ver.2 | total |
---|---|---|---|
5 | 5 | 5 | 15 |
pspA | pSB1C3 | Ligation High Ver.2 | total |
---|---|---|---|
4 | 1 | 3 | 8 |
We did these for an hour at 16℃.
- pspA (5µL)→377fmol
- DT→39fmol
- pspA (4µL)→339fmol
- pSB1C3→34fmol
Transformation
pspA-DT, pspA (pSB1C3), torA (pSB1C3) and GFP-DT
March 20
Screaning PCR
Quick Taq | Primer-R | Primer-F | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
- pspA→○
- pspA-DT→○
- GFP-DT→○
- torA→×
- Kil-DT 6 of 8 sumples→○
Quick Taq | VR | VF | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
- pspA→○
- pspA-DT→×
Restrictive Digestion
torA | EcoR1 | Spe1 | BSA | BufferM | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.3 | 0.3 | 0.3 | 3 | 16.1 | 30 |
DT | EcoR1 | Xba1 | BSA | BufferM | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.2 | 0.2 | 0.3 | 3 | 16.3 | 30 |
We did this for overnight at 37℃. And we did purification.
torA 34.2ng/µL
DT 28.0ng/µL
March 21
Miniprep
GFP-DT-1 : 77.7ng/µL
GFP-DT-2 : 67.6ng/µL
Restrictive Digestion
pSB1C3 | EcoR1 | Spe1 | BSA | BufferM | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.2 | 0.2 | 0.3 | 3 | 16.3 | 30 |
5 | 0.2 | 0 | 0.2 | 2 | 12.6 | 20 |
We did this for 3 hours at 37℃, and then we did gel extraction. We got 25.1ng/µL pSB1C3
GFP-DT | EcoR1 | Pst1 | Xba1 | BSA | BufferM | MilliQ | total |
---|---|---|---|---|---|---|---|
5 | 0.2 | 0.2 | 0 | 0.2 | 2 | 12.4 | 20 |
5 | 0.2 | 0 | 0 | 0.2 | 2 | 12.6 | 20 |
10 | 0.2 | 0 | 0.2 | 0.3 | 3 | 16.3 | 30 |
We did this for 3 hours at 37℃, and then we did Purification. We got 30.7ng/µL GFP-DT.
Ligation
torA | pSB1C3 | pspA | DT | GFP-DT | Ligation High Ver.2 | total | |
---|---|---|---|---|---|---|---|
1 | 4 | 1 | 0 | 0 | 0 | 3 | 8 |
2 | 0 | 1 | 7 | 0 | 0 | 4 | 12 |
3 | 0 | 0 | 5 | 3 | 0 | 4 | 12 |
4 | 3 | 0 | 0 | 0 | 5 | 4 | 12 |
We did this for an hour at16℃.
March 22
PCR
We did PCR to amplify torA_signal that was product of PCR at March 5 with redesigned primers.
Buffer | dNTPs | MgSO4 | Primer-F | Primer-R | Template | MilliQ | KOD plus neo | total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 0.5 | 32.5 | 1 | 50 |
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 10sec
→30cycles
→Purification 110.7ng/µL
Restrictive Digestion
torA | EcoR1 | Spe1 | BSA | BufferM | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.2 | 0.2 | 0.3 | 3 | 16.3 | 30 |
Ligation
torA | pSSB1C3 | pspA | DT | GFP-DT | Ligation high | total | |
---|---|---|---|---|---|---|---|
1 | 4 | 3 | 0 | 0 | 0 | 4 | 11 |
2 | 3 | 0 | 0 | 0 | 3 | 3 | 9 |
3 | 0 | 0 | 5 | 5 | 0 | 5 | 15 |
- torA (4µL)→512fmol
- pSB1C3→54fmol
- torA (3µL)→384fmol
- GFP-DT→36fmol
- pspA→377fmol
- DT→65fmol
March 23
Screening PCR
torA (pSB1C3), torA-GFP-DT and pspA-DT
Quick Taq | VF | VR | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
E.coli in liquid culture that had pspA(pSB1C3) also expressed RFP.
Restrictive Digestion
pspA | EcoR1 | Spe1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
5 | 0.3 | 0.3 | 3 | 0.3 | 21.1 | 30 |
And then we did ethanol precipitation
Ethanol precipitation
pspA 11.5ng/µL.
Miniprep
LacP+pSB3C5-8 77.9µg/mL
LacP+pSB3C5-10 69.0µg/mL
Kil+DT-4 58.0µg/mL
Kil+DT-7 15.2µg/mL
Restrictive Digestion
LacP+pSB3C5-8 | Spe1 | Pst1 | Buffer2 | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
Kil+DT-4 | Xba1 | Pst1 | Buffer2 | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
at 37℃ for 1.5 hours
And then we did Gel extraction.
Gel Extraction
LacP+pSB3C5-8 40.4µg/mL
Kil+DT-4 26.8µg/mL
March 26
Miniprep
torA(pSB1C3) 18.5[ng/µL]
torA-GFP-DT 20.0[ng/µL]
Restrictive Digestion
torA(pSB1C3) | EcoR1 | Pst1 | BufferH | BSA | MilliQ | total |
---|---|---|---|---|---|---|
5 | 0.2 | 0.2 | 2 | 0.2 | 12.4 | 20 |
5 | 0.2 | 0 | 2 | 0.2 | 12.6 | 20 |
torA-GFP-DT | EcoR1 | Xba1 | Pst1 | BufferH | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|---|---|
5 | 0.2 | 0 | 0.2 | 2 | 0 | 0.2 | 12.4 | 20 |
5 | 0.2 | 0 | 0 | 2 | 0 | 0.2 | 12.6 | 20 |
20 | 0 | 0.2 | 0.2 | 0 | 3 | 0.3 | 6.3 | 30 |
We did Gel extraction and then got ??? 28.7[ng/µL]
Ligation
LacP (pSB3C5) | torA-GFP-DT | Ligation High Ver.2 | total |
---|---|---|---|
1 | 5 | 3 | 9 |
- LacP : 22fmol
- torA-GFP-DT : 197fmol
pspA | pSB1C3 | Ligation High Ver.2 | total |
---|---|---|---|
10 | 1 | 5 | 16 |
pspA | DT | Ligation High Ver.2 | total |
---|---|---|---|
10 | 1 | 5 | 16 |
- pspA : 180fmol
- pSB1C3 : 18fmol
- DT : 16fmol
LacP(pSB3C5) | Kil-DT | Ligation High Ver.2 | total |
---|---|---|---|
1 | 5 | 3 | 9 |
for 2 hours at 16℃
Transformation
LacP-Kil-DT | competent cell | total |
---|---|---|
1 | 10 | 11 |
March 27
Miniprep
We retryed miniprep of torA(pSB1C3).
We got torA and its concentration was 39.3[ng/µL].
Transformation
Name | Well | Sample | Competent Cells | Total | Plate | Colony |
---|---|---|---|---|---|---|
BBa_K117004 | 14J(2011 plate2) | 5 | 20 | ? | ? | ? |
We added 100[µL] of culture medium before we started culturing the E.coli.
Screening PCR
Quick Taq | VF2 | VR | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
LacP-torA-GFP-DT 1, 3, 5, 6, 8, 9 was successful.
pspA-DT was failed.
E.coli that had pspA(pSB1C3) did not make any colony.
LacP-Kil-DT 1,2,3,4,5,6,7,8 was failed.
Liquid culture
LacP-torA-GFP-DT
July 23
Transformation
DT(64.4ng/μl) | Competent cell | Plating in SOC medium | Total |
---|---|---|---|
1 | 20 | 100 | 121 |
July 24
Plating in SOC medium
July 25
Transformation
- BBa_J23113 from iGEM Kit
- LacP from iGEM Kit
July 25
July 26
July 27
Restrictive Digestion
Kil +DT (44.0 ng/μl) | 10xBuffer2 | BSA | Xbal | MilliQ | Total |
---|---|---|---|---|---|
13.6 | 3.0 | 0.5 | 0.5 | 11.9 | 30.0 |
LacP(28.7ng/μl) | 10xBuffer2 | BSA | Spe1 | Pst1 | Total |
---|---|---|---|---|---|
20.9 | 3.0 | 0.5 | 0.5 | 4.6 | 30.0 |
July 30
Ligation
kil+DT(Xba1,Pst1) | LacP(Spe1,Pst1) | Ligation High | Total |
---|---|---|---|
30 | 6 | 36 | 72 |
July 31
Restriction Enzyme Processing
torA-GFP+DT | 10×M Buffer | BSA | MilliQ | Xba1 | Pst1 | Total |
---|---|---|---|---|---|---|
2.5 | 3.0 | 0.5 | 23.4 | 0.3 | 0.3 | 30.0 |
Liquid Culture
J23113(backbone J61002)
August 1
Restrictive Digestion
LacP+kil+DT | BSA | 10×H Buffer | EcoR1 | Pst1 | MilliQ | Total |
---|---|---|---|---|---|---|
5.0 | 0.5 | 3.0 | 0.5 | 0.5 | 20.5 | 30.0 |
LacP middle copy | BSA | 10×H Buffer | EcoR1 | Pst1 | MilliQ | Total |
---|---|---|---|---|---|---|
10.0 | 0.5 | 3.0 | 0.5 | 0.5 | 15.5 | 30.0 |
MIniprep
J23113(backbone J61002)
Liquid culture
J23113(backboneJ61002) Three test tubes of 4 mL LB medium with ampicillin 37℃ overnight
August 2
August 3
Restrictive Digestion
pSB4K5 | BSA | 10×H Buffer | EcoR1 | Pst1 | MilliQ | Total |
---|---|---|---|---|---|---|
8.0 | 0.5 | 3.0 | 0.5 | 0.5 | 17.5 | 30.0 |
B0034 | BSA | 10×H Buffer | Spe1 | Pst1 | MilliQ | Total |
---|---|---|---|---|---|---|
12.0 | 0.5 | 3.0 | 0.5 | 0.5 | 13.5 | 30.0 |
37℃ 2h
DNA purification
Ligation
LacP+kil+DT | pSB4K5 | ligation high | Total |
---|---|---|---|
15 | 15 | 15 | 45 |
Miniprep
- J23113(backbone J61002) 218.0μg/mL
- J23113(backbone J61002) 252.5μg/mL
- J23113(backbone J61002) 201.0μg/mL
- pSB3C5 45.0μg/ml 1.60 260/280 1.80 260/230
- pSB4C5 213.0μg/mL 1.77 260/280 4.40 260/230
August 4
August 5
August 6
August 7
August 8
August 9
August 10
Ligation
B0034 Spe1 Pst1 | GFP+DT Xba1 Pst1 | ligation high | Total |
---|---|---|---|
2 | 3 | 4 | 9 |
Transformation
- RBS+GFP+DT
- LacP+kil+DT
- RBS(for control)
- To put 2ng DNA in 20μL competent cell and leave it on ice for 30min
- Heat shock for 60s at 42℃
- To leave on ice for 2min
- To spread on ampicillin LB plate
August 11
August 12
August 13
Liquid culture
B0034 3mL ×3
- 37℃ overnight
August 14
Miniprep
- B0034 0μg/mL
- B0034 235.5μg/mL
- B0034 76.5μg/mL
Colony PCR
LacP+kil+DT ×5
2×Quick Taq | VF | VF | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
25cycles
pspA
10×Buffer for KOD-Plus-Ver2 | 2mM dNTPs | 25mM MgSO4 | Primer PsPA f-p | Primer PsPA r-s | KOD-Plus- | MilliQ | Total |
---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |
August 15
Colony PCR
pspA
2×Quick Taq | pspA r-s | pspA f-p | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃, 2min
94℃, 30sec
56℃, 30sec
68℃, 1min
→35cycles
August 16
Electrophoresis
positive control(GFP+DT)
negative control(MilliQ)
① ~⑥ colony PCR LacP+kil+DT
①1kb ladder
②Positive control (GFP+DT)
③Negative control (MilliQ)
④~⑨LacP+kil+DT ①~⑥
⑩~⑫pspA 10μL ,6×buffer 2μL
⑬1kb ladder
August 17
Colony PCR
pspA
10×Buffer for KOD-Plus-Ver2 | 2mM dNTPs | 25mM MgSO4 | Primer PsPA f | Primer PsPA r | KOD-Plus- | MilliQ | Total |
---|---|---|---|---|---|---|---|
5 | 3 | 3 | 1.5 | 1.5 | 1 | 35 | 50 |
5 | 3 | 5 | 1.5 | 1.5 | 1 | 33 | 50 |
negative control(MilliQ 50μL)
94℃, 2min
94℃, 15sec
55℃, 30sec
68℃, 1min
→35cycles
Electrophoresis
①1kb ladder
②, ③ MgSO4 3μL, pspA
④, ⑤ MgSO4 5μL, pspA
⑥ negative control(MilliQ)
⑧ 1kb ladder
August 18
August 19
August 20
Restrictive Digestion
J23113(201.0ng/μL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 1 | 1 | 3 | 0.5 | 14.5 | 30 |
August 21
Colony PCR
pspA
10×Buffer | 2mM dNTPs | 25mM MgSO4 | Primer PsPA f | Primer PsPA r | KOD Plus Neo | MilliQ | Total |
---|---|---|---|---|---|---|---|
5 | 3 | 5 | 1.5 | 1.5 | 1 | 33 | 50 |
5 | 3 | 7 | 1.5 | 1.5 | 1 | 31 | 50 |
5 | 3 | 10 | 1.5 | 1.5 | 1 | 28 | 50 |
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→25cycles
Restrictive Digestion
J23113(218ng/μL) | Spe1 | Pst1 | BufferH | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 1 | 1 | 3 | 0.5 | 14.5 | 30 |
torA-GFP-DT(20ng/μL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
5 | 0.6 | 0.6 | 3 | 0.5 | 20.3 | 30 |
Electrophoresis
① 1kb ladder
②, ③ torA-GFP-DT(Xba1,Pst1)
④, ⑤ J23113(Spe1,Pst1)
Transformation
- LacP-torA-GFP-DT(Backbone pSB3C5)
- BBa_K11704(Backbone pSB1A2)
Electrophoresis
laneA-C. pspA
laneD. pspA(without primer)
Colony PCR
- pspA
10×Buffer | 2mM dNTPs | 25mM MgSO4 | Primer PsPA f | Primer PsPA r | KOD Plus Neo | MilliQ | Total |
---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |
- negative control for pspA
10×Buffer | 2mM dNTPs | 25mM MgSO4 | Primer PsPA f-p | Primer PsPA r-s | KOD Plus Neo | MilliQ | Total |
---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |
- GFP-DT(positive control)
10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | Total |
---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
Transformation
- LacP-torA-GFP-DT(Backbone pSB3C5)
- J23107-tatABCD
PCR
kil, torA-GFP
10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | DNA | Total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50 |
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→25cycles
August 22
Restrictive Digestion
pSB3C5 | EcoR1 | Pst1 | BufferH | BSA | MilliQ | total |
---|---|---|---|---|---|---|
13.3 | 0.5 | 0.5 | 3 | 0.5 | 12.2 | 30 |
Gel extraction
pSB3C5(EcoR1, Pst1)
27.2μg/mL
Transformation(the second time)
- LacP-torA-GFP-DT(Backbone pSB3C5)
- J23107-tatABCD
Electrophoresis
See "August 21 -Colony PCR-"
1. 1kb ladder
2. pspA(Primer pspA f&r)
3. negative control for 2
4. GFP-DT
5. pspA(Primer pspA f-p&r-s)
6. pspA(Primer pspA f-p&r-s)
7. negative control for 5&6
PCR
LacP-GFP-DT(a kit of biological parts)
10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | DNA | Total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50 |
Colony PCR
pspA
10×Buffer | 2mM dNTPs | 25mM MgSO4 | Primer pspA f | Primer pspA r | KOD Plus Neo | MilliQ | Total |
---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |
10×Buffer | 2mM dNTPs | 25mM MgSO4 | Primer pspA f-p | Primer pspA r-s | KOD Plus Neo | MilliQ | Total |
---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→30cycles
Electrophoresis
1. 1kb ladder
2. pspA(Primer pspA f&r)
3. pspA(Primer pspA f-p&r-s)
4. negative control for pspA(Primer pspA f&r)
5. negative control for pspA(Primer pspA f-p&r-s)
Transformation
Kil
August 23
Colony PCR
J23107-tatABCD
VF and VR were used for amplifying approximately 2800bp
10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | DNA | Total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 32.5 | 0.5 | 50 |
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 90sec
→25cycles
August 24
Purification of PCR products
pspA(Primer pspA f-p&r-s)
84.1μg/mL
→See "August 22 -Colony PCR-"
Restrictive Digestion
pspA | EcoR1 | Pst1 | BufferH | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
pspA | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
at 37℃ for 1h
Gel extraction
- pspA(EcoR1, Pst1) 30.7μg/mL
- pspA(Xba1, Pst1) 44.0μg/mL
Restrictive Digestion
pSB1C3(82.9μg/mL) | EcoR1 | Pst1 | BufferH | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
→Gel extraction
Electrophoresis
1. 1kb ladder
2. J23107-tatABCD →See "August 23 -PCR-"
3. negative control for J23107-tatABCD
4. pSB1C3(EcoR1, Pst1)
Ligation
pSB3C5(EcoR1, Pst1) | pspA(EcoR1, Pst1) | Ligation High Ver.2 | total |
---|---|---|---|
1 | 5 | 3 | 9 |
16℃, overnight
August 25
August 26
August 27
Ligation
pSB1C3(EcoR1, Pst1) | pspA(EcoR1, Pst1) | Ligation High Ver.2 | total |
---|---|---|---|
1 | 5 | 3 | 9 |
16℃, overnight
Colony PCR
Kil (1-7)
10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | Total |
---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→30cycles
Purification of PCR products
torA-GFP →See "August 21 -PCR-"
Restrictive Digestion
LacP(Backbone:pSB3C5) | Spe1 | Pst1 | BufferH | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
at 37℃ for 1h
→Gel extraction
torA-GFP | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
at 37℃ for 1h
→Purification
Purification of PCR products
J23107-tatABCD
42.0μg/mL
→See "August 23 -PCR-"
Restrictive Digestion
J23107-tatABCD | Spe1 | EcoR1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
August 28
Colony PCR
Kil (1-16)
10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | Total |
---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |
Transformation
- pspA-pSB1C3
- pspA-pSB3C5
Liquid culture
- Kil-3 →Miniprep 100.4μg/mL
- Kil-6
- Kil-7 →Miniprep 77.7μg/mL
- LacP-torA-GFP-1 →Miniprep 88.3μg/mL
- LacP-torA-GFP-2 →Miniprep 85.0μg/mL
- LacP-torA-GFP-3 →Miniprep +++
August 29
Kil assay
After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600.
IPTG | 0 | 0.001mM | 0.01mM | 0.1mM | 1mM |
---|---|---|---|---|---|
0.5h | 0.202 | 0.207 | 0.201 | 0.207 | 0.200 |
1h | 0.406 | 0.428 | 0.424 | 0.402 | 0.421 |
1.5h | 0.796 | 0.813 | 0.779 | 0.751 | 0.802 |
2h | 1.107 | 1.129 | 1.141 | 1.092 | 1.124 |
2.5h | 1.546 | 1.565 | 1.541 | 1.532 | 1.578 |
3h | 1.912 | 1.933 | 1.890 | 1.883 | 1.940 |
3.5h | 2.300 | 2.295 | 2.238 | 2.247 | 2.259 |
4h | 2.546 | 2.545 | 2.528 | 2.490 | 2.520 |
4.5h | 2.688 | 2.663 | 2.603 | 2.633 | 2.666 |
5h | 2.699 | 2.826 | 2.787 | 2.593 | 2.673 |
5.5h | 2.863 | 2.742 | 2.754 | 2.741 | 2.756 |
6h | 2.831 | 2.876 | 2.758 | 2.759 | 2.728 |
20h | 2.671 | 2.706 | 2.680 | 2.606 | 2.619 |
Liquid culture
- pspA-pSB1C3(1-3)
- pspA-pSB3C5(1-3)
Restrictive Digestion
LacP-Kil-DT(134.7ng/μL) | EcoR1 | Spe1 | BufferM | MilliQ | total |
---|---|---|---|---|---|
10 | 1 | 1 | 3 | 15 | 30 |
37℃, overnight
Colony PCR
- LacP-torA-GFP-DT (1-5)
- pspA-pSB1C3 (1-5)
- pspA-pSB3C5 (1-6)
10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | Total |
---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 40sec
→30cycles
Gel extraction
J23107-tatABCD(EcoR1, Pst1) -5.9μg/mL
August 30
Ligation
J23107-tatABCD(EcoR1, Spe1)22.2μg/mL | DT(EcoR1, Xba1)37.5μg/mL | Ligation High Ver.2 | total |
---|---|---|---|
10 | 1 | 9 | 20 |
at 16℃ for 1h
Transformation
J23107-tatABCD-DT (Amp resistance)
Electrophoresis
1. LacP-torA-GFP-DT-1
2. LacP-torA-GFP-DT-2
3. LacP-torA-GFP-DT-3
4. LacP-torA-GFP-DT-4
5. LacP-torA-GFP-DT-5
6. pspA-pSB1C3-1
7. pspA-pSB1C3-2
8. pspA-pSB1C3-3
9. pspA-pSB1C3-4
10.pspA-pSB1C3-5
11.pspA-pSB3C5-1
12.pspA-pSB3C5-2
13.pspA-pSB3C5-3
14.pspA-pSB3C5-4
15.pspA-pSB3C5-5
16.pspA-pSB3C5-6
Miniprep
- pspA-pSB3C5-1 176μg/mL
- pspA-pSB3C5-2 214μg/mL
- pspA-pSB3C5-3 461μg/mL
- pspA-pSB1C3-2 79μg/mL
Colony PCR
LacP-torA-GFP-DT (6-13)
2×Quick Taq | VF | VR | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃, 2min
94℃, 30sec
56℃, 30sec
68℃, 1min
→30cycles
Restrictive Digestion
pspA-pSB1C3 | EcoR1 | Spe1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
15 | 0.5 | 0.5 | 5 | 0.5 | 28.5 | 50 |
at 37℃ for 1h
Electrophoresis
1. 1kb ladder
3. pspA-pSB1C3(EcoR1, Spe1)
August 31
Gel extraction
pspA-pSB1C3(EcoR1, Spe1) 8.0μg/mL
Purification
LacP-GFP-DT -0.7μg/mL
Ligation
pspA(EcoR1, Spe1) | DT(EcoR1, Xba1) | Ligation High Ver.2 | total |
---|---|---|---|
25 | 1 | 13 | 39 |
at 16℃ for 1h
Transformation
pspA-DT (Amp resistance)
September 1
September 2
Colony PCR
J23107-tatABCD-DT (1-8)
2×Quick Taq | VF | VR | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 3min
→25cycles
pspA-DT (2-8)
2×Quick Taq | VF | VR | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
Extension, 10sec
→25cycles
Restrictive Digestion
LacP-torA-GFP-DT | EcoR1 | Spe1 | BufferH | MilliQ | total |
---|---|---|---|---|---|
10 | 1 | 1 | 2 | 6 | 20 |
at 4℃ for overnight
Electrophoresis
1. 1kb ladder
3. J23107-tatABCD-DT-1
4. J23107-tatABCD-DT-3
5. J23107-tatABCD-DT-4
6. J23107-tatABCD-DT-5
7. J23107-tatABCD-DT-6
8. J23107-tatABCD-DT-7
9. J23107-tatABCD-DT-8
1. 1kb ladder
3. J23107-tatABCD-DT-1
4. J23107-tatABCD-DT-3
5. J23107-tatABCD-DT-4
6. J23107-tatABCD-DT-5
7. J23107-tatABCD-DT-6
8. J23107-tatABCD-DT-7
9. J23107-tatABCD-DT-8
11. LacP-pSB3C5(Spe1, Pst1)
1. 1kb ladder
3. pspA-DT-2
4. pspA-DT-3
5. pspA-DT-4
6. pspA-DT-5
7. pspA-DT-6
8. pspA-DT-7
9. pspA-DT-8
September 3
PCR
tatABCD-DT
10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | DNA | Total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50 |
Gel extraction
LacP-torA-GFP-DT →See "September 5 -Gel extraction-"
Restrictive Digestion
torA-GFP-DT | Xba1 | Pst1 | BufferM | MilliQ | total |
---|---|---|---|---|---|
20 | 2 | 2 | 4 | 12 | 40 |
Purification
J23107-tatABCD 29.6μg/mL 1.56(260/280) 1.60(260/230)
Electrophoresis
1. 1kb ladder
2. J23107-tatABCD
Liquid culture
- LacP-kil-DT(1)
- pspA-DT(4, 7)
PCR
J23107-tatABCD
10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | DNA | Total |
---|---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 29 | 4 | 50 |
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 10sec
→30cycles
Gel extraction
LacP-torA-GFP-DT 31μg/mL 1.28(260/280) 0.49(260/230) LacP-torA-GFP-DT 8μg/mL 1.32(260/280) 0.83(260/230)
Transformation
J23107-tatABCD (Amp resistance)
J23113-kil-DT (Amp resistance)
J23100-kil-DT (CP resistance)
J23101-kil-DT (CP resistance)
J23106-kil-DT (CP resistance)
J23115-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.
J23105-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.
Electrophoresis
1. 1kb ladder
2-7. LacP-torA-GFP-DT
Colony PCR
LacP-torA-GFP-DT (14-21) J23113-kil-DT (Amp resistance, 1-4) J23113-kil-DT (CP resistance, 1-4)
2×Quick Taq | primer-f | primer-r | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1min
→25cycles
September 4
Liquid culture
- LacP-kil-DT(4, 5)(Amp resistance)
- pspA-DT(3, 6, 7)(Amp resistance)
PCR
We did PCR 10 more cycles to amplify products of PCR that we had done yesterday because we could not amplify J23107-tatABCD on September 3.
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 1.5min
→30cycles
Electrophoresis
1-8. LacP-torA-GFP-DT
1-4. J23113-kil-DT (Amp resistance)
5-8. J23113-kil-DT (CP resistance)
Liquid culture
- LacP-torA-GFP-DT
- J23113-kil-DT
- LacP-kil-DT(with IPTG)
- LacP-kil-DT(without IPTG)
After 20 hour, we quantified OD600.
plasmid | OD600 |
---|---|
LacP-kil-DT(with IPTG) | 2.116 |
LacP-kil-DT(without IPTG) | 2.320 |
We cultured CP tolerance plasmid and give an antibiotic(Amp or Kan or none) after 2 hours. We quantify OD600.
time | 1 | 2 | 3 |
---|---|---|---|
2hours | 0.131 | 0.100 | 0.606 |
add an antibiotic | Amp | Kan | none |
7hours | 0.491 | 0.988 | 2.634 |
19hours | 2.253 | 0.815 | 2.742 |
September 5
Colony PCR
J23107-tatABCD (1-7)
buffer | 2mM dNTPs | 25mM MgSO4 | primer-f | primer-r | KOD Plus Neo | DW | Total |
---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 1.25min
→30cycles
Gel extraction
by Kato
See "September 3 -Gel extraction-"
LacP-torA-GFP-DT 15.9μg/mL 1.29(260/280) 0.38(260/230)
LacP-torA-GFP-DT 17.2μg/mL 1.23(260/280) 0.48(260/230)
Miniprep
- J23113-kil-DT 85.7μg/mL 1.76(260/280) 1.97(260/230)
- LacP-torA-GFP-DT 126.6μg/mL 1.81(260/280) 2.09(260/230)
- LacP-kil-DT-4 62.5μg/mL 1.79(260/280) 1.99(260/230)
- LacP-kil-DT-5 56.0μg/mL 1.79(260/280) 2.01(260/230)
- pspA-DT-3 322μg/mL 1.45(260/280) 1.42(260/230)
- pspA-DT-6 147.6μg/mL 1.25(260/280) 1.37(260/230)
- pspA-DT-7 145.5μg/mL 1.24(260/280) 1.27(260/230)
Restrictive Digestion
pspA-DT-6 | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
at 37℃ for 1h
Electrophoresis
J23107-tatABCD (1-7)
Colony PCR
J23107-tatABCD (8-23)
2×Quick Taq | VF2 | VR | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1.5min
→30cycles
Gel extraction
pspA-DT 10.2μg/mL 1.42(260/280) 0.76(260/230)
Ligation
pspA-DT(Xba1, Pst1) | LacP(pSB3C5)(Spe1, Pst1) | Ligation High Ver.2 | total |
---|---|---|---|
5 | 1 | 3 | 9 |
at 16℃
Electrophoresis
LacP-torA-GFP-DT(8-15)
LacP-torA-GFP-DT(9-23)
Ligation
LacP-torA-GFP-DT(EcoR1, Spe1) | DT 17.2μg/mL(Xba1, Pst1) | Ligation High Ver.2 | total |
---|---|---|---|
15 | 25 | 20 | 60 |
Liquid culture
- J23107-tatABCD-8
- J23107-tatABCD-20
- LacP-torA-GFP-DT-21
September 6
Miniprep
- J23107-tatABCD-8 82.4μg/mL 2.06(260/280) 2.95(260/230)
- J23107-tatABCD-20 102.9μg/mL 2.04(260/280) 2.94(260/230)
Restrictive Digestion
J23107-tatABCD 102.9μg/mL | Spe1 | Pst1 | BufferH | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
at 37℃ for 1h
Transformation
LacP-pspA-DT(pSB3C5)
DNA | Competent cell | total |
---|---|---|
2 | 20 | 22 |
Gel extraction
J23107-tatABCD(Spe1, Pst1) →We failed gel extraction because we mistook steps of experiment.
Electrophoresis
LacP-torA-GFP-DT-pspA-DT
Restrictive Digestion
by Terasaka
J23107-tatABCD | Spe1 | Pst1 | BufferH | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
Gel extraction
J23107-tatABCD(Spe1, Pst1) →See "September 7 -Gel extraction-"
September 7
Liquid culture
- LacP-torA-GFP-DT-1 (CP resistance)
- LacP-torA-GFP-DT-21 (CP resistance)
preculture for 1 hour
Colony PCR
LacP-torA-GFP-DT (1-8) LacP-pspA-DT (1-8)
2×Quick Taq | VF2 | VR | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1.5min
→30cycles
Restrictive Digestion
by Terasaka
J23107-tatABCD | EcoR1 | Spe1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
GFP-DT | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
15 | 0.5 | 0.5 | 3 | 0.5 | 10.5 | 30 |
Electrophoresis
① -⑧. LacP-pspA-DT
1-8. LacP-torA-GFP-DT
Gel extraction
- J23107-tatABCD(EcoR1, Spe1)
- J23107-tatABCD(Pst1, Spe1) ①57.6μg/mL ②23.4μg/mL
- GFP-DT(Xba1, Pst1)
Restrictive Digestion
pspA-DT(145.5μg/mL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
37℃, overnight
Liquid culture
- LacP-torA-GFP-DT-7 (LB 2mL)
+IPTG 0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM, 0.3mM
- LacP-torA-GFP-DT-7 (LB 5mL)
September 8
Check the effect of Amp
We used 5mL of the liquid culture medium with LacP-torA-GFP-DT-7.
→See "September 7 -Liquid culture-"
We diluted it with LB until OD600=0.444, and dispensed it.
The dispense volume was 1.3mL.
We added 0/13/130μL of Amp(50mg/mL) to it.
5.5h after incubating at 37℃
Amp | 0μL | 13μL | 130μL |
---|---|---|---|
OD600 | 2.457 | 2.696 | 0.310 |
Observation through a confocal microscope
We used the liquid culture media with LacP-torA-GFP-DT + 13μL of Amp(50mg/mL) + IPTG 0/0.1/0.15mM.
Their OD600 was 0.47.
3h after incubating at 37℃, we eliminated each supernatant using a centrifuge, and diluted them with LB to which Amp was added.
2.5h after incubating at 37℃, we observed them through a confocal microscope.
We failed to observe it.
Gel extraction
pspA-DT -5.5μg/mL
Restrictive Digestion
pspA-DT-3(322μg/mL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 1 | 1 | 3 | 3 | 12 | 30 |
at 37℃ for 1h
September 9
Restrictive Digestion
J23107-tatABCD(102.9μg/mL) | EcoR1 | Spe1 | BufferM | MilliQ | total |
---|---|---|---|---|---|
10 | 1 | 1 | 3 | 15 | 30 |
Electrophoresis
1. J23107-tatABCD(EcoR1, Spe1)
2. J23107-tatABCD(before restriction)
3. pspA-DT(Xba1, Pst1)
4. pspA-DT(before restriction)
Gel extraction
- J23107-tatABCD(EcoR1, Spe1) 26μg/mL
- pspA-DT(Xba1, Pst1) 199μg/mL
Liquid culture
- J23100/J23101/J23106-1
- pspA-DT-5 ×2
- LacP-torA-GFP-DT-21
- GFP generator-2
- LacP-pspA-DT-6
September 10
Miniprep
- J23100-1 105.5μg/mL
- J23101-1 76.0μg/mL
- J23106-1 90.9μg/mL
- pspA-DT-5 ①104.1μg/mL ②80.0μg/mL
- LacP-torA-GFP-DT-21 79.6μg/mL
- GFP generator-2 84.1μg/mL
- LacP-pspA-DT-6 99.8μg/mL
Ligation
- J23107-tatABCD-DT
J23107-tatABCD(EcoR1, Spe1) | DT(EcoR1, Xba1) | Ligation High Ver.2 | total |
---|---|---|---|
10 | 5 | 7.5 | 22.5 |
- Negative control for J23107-tatABCD-DT
DT(EcoR1, Xba1) | Ligation High Ver.2 | total |
---|---|---|
5 | 5 | 10 |
- J23107-tatABCD-pspA-DT
J23107-tatABCD(Spe1, Pst1) | pspA-DT(Xba1, Pst1) | Ligation High Ver.2 | total |
---|---|---|---|
3 | 6 | 9 | 18 |
- Negative control for J23107-tatABCD-pspA-DT
J23107-tatABCD(Spe1, Pst1) | Ligation High Ver.2 | total |
---|---|---|
3 | 3 | 6 |
Restrictive Digestion
DNA | Spe1 | Pst1 | BufferH | MilliQ | total |
---|---|---|---|---|---|
15 | 1 | 1 | 3 | 10 | 30 |
DNA=J23100(105.5μg/mL), J23101(76.0μg/mL), J23106(90.9μg/mL)
at 37℃
Gel extraction
J23100 27.1μg/mL
J23101 50.2μg/mL
J23106 16.1μg/mL
Ligation
- J23100/J23101/J23106-kil-DT
J23100/J23101/J23106(Spe1, Pst1) | kil-DT(Xba1, Pst1) | Ligation High Ver.2 | total |
---|---|---|---|
1 | 5 | 3 | 9 |
- Negative control for J23100/J23101/J23106-kil-DT
J23100/J23101/J23106(Spe1, Pst1) | MilliQ | Ligation High Ver.2 | total |
---|---|---|---|
1 | 5 | 3 | 9 |
Transformation
J23100/J23101/J23106-kil-DT
September 11
Colony PCR
- J23107-tatABCD-DT (1-8)
- J23107-tatABCD-pspA-DT (1-8)
2×Quick Taq | VF2 | VR | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 3.5min
→30cycles
Electrophoresis
- J23107-tatABCD-DT (1-8)
- J23107-tatABCD-pspA-DT (1-8)
Restrictive Digestion
GFP generator(84.1μg/mL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
15 | 1 | 1 | 3 | 3 | 7 | 30 |
LacP-torA-GFP-DT(85.0μg/mL) | Spe1 | Pst1 | BufferH | MilliQ | total |
---|---|---|---|---|---|
15 | 1 | 1 | 3 | 10 | 30 |
LacP-pspA-DT(99.8μg/mL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
15 | 1 | 1 | 3 | 3 | 7 | 30 |
Liquid culture
pSB1C3(self-ligation) ×5 in LB 3mLCP 3μL
When OD600=approximately 0.6, we added 0/30/60/150/300μL of Amp(50mg/mL) to each liquid culture medium.
1h and 5h after incubating at 37℃, we measured OD600.
1h | 5h | |
---|---|---|
0 | -0.020 | 0.403 |
30 | -0.046 | 0.544 |
60 | 0.263 | 0.007 |
150 | 0.061 | 0.839 |
300 | 0.093 | 1.441 |
Ligation
pSB3C5(EcoR1, Pst1) | pspA-DT(Xba1, Pst1) | J23107-tatABCD(EcoR1, Spe1) | Ligation High Ver.2 | total |
---|---|---|---|---|
1 | 3 | 5 | 4.5 | 13.5 |
4℃, overnight
Colony PCR
- J23100-kil-DT (1-6)
- J23101-kil-DT (1-7)
- J23106-kil-DT (1-2)
2×Quick Taq | VF | VR | MilliQ | Total |
---|---|---|---|---|
23 | 1 | 1 | 25 | 50 |
Gel extraction
- GFP generator (Xba1, Pst1) 14.1μg/mL
- LacP-torA-GFP-DT (Spe1, Pst1) 42.9μg/mL 1.13(260/280) 1.19(260/230)
- LacP-pspA-DT (Xba1, Pst1) 20.0μg/mL 1.01(260/280) 1.30(260/230)
Electrophoresis
- J23107-tatABCD-DT (1-8)
- J23106-kil-DT (1, 2)
- J23100-kil-DT (1-4)
Liquid culture
- LacP-torA-GFP-DT
- pSB1C3
Colony PCR
We did PCR 10 more cycle to amplify products of PCR on 9/11 of J23107-tatABCD-pspA-DT and J23107-tatABCD-DT.
September 12
Colony PCR
J23107-tatABCD-pspA-DT (9-15)
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 4min
→26cycles
Check the effect of Amp
We cultured pSB1C3 for overnight.
→See "September 7 -Liquid culture-"
We diluted it with LB, and dispensed it.
We added Amp(50mg/mL) to it until density of Amp was 1/10, 1/30, 1/50 .
Amp | start | 5 hours |
---|---|---|
1/10 | 0.557 | 0.077 |
1/30 | 0.628 | 0.166 |
1/50 | 0.472 | 0.055 |
0 | 0.594 | 2.585 |
Electrophoresis
- J23107-tatABCD-pspA-DT
- J23101-kil-DT (1-7)
Restrictive Digestion
pspA(pSB3C5) (106μg/mL) | EcoR1 | Pst1 | BufferH | MilliQ | total |
---|---|---|---|---|---|
15 | 1 | 1 | 3 | 10 | 30 |
pspA-DT-3 | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
15 | 1 | 1 | 3 | 3 | 7 | 30 |
J23107-tatABCD (102.4μg/mL) | EcoR1 | Spe1 | BufferM | MilliQ | total |
---|---|---|---|---|---|
15 | 1 | 1 | 3 | 10 | 30 |
Electrophoresis
- pspA(pSB3C5)(EcoR1, Pst1)
- pspA-DT(Xba1, Pst1)
- J23107-tatABCD(EcoR1, Spe1)
- pSB1C3(EcoR1, Spe1)
Transformation
J23107-tatABCD-pspA-DT(pSB3C5)
DNA | Competent cell | total |
---|---|---|
2 | 20 | 22 |
on ice for 30 minites heatshock at 42℃ for 1 hour on ice 2 minites then add SOC medium 200μl preculture at 37℃ for 1 hour incubate CP culture plate
Gel extraction
- pspA(pSB3C5)(EcoR1, Pst1) 26.8μg/mL 1.27(260/280) 0.32(260/230)
- pspA-DT(Xba1, Pst1) 26.1μg/mL 1.13(260/280) 0.55(260/230)
- J23107-tatABCD(EcoR1, Spe1) 1.5μg/mL 1.99(260/280) 0.06(260/230)
Ligation
pSB3C5(EcoR1, Pst1) 26.8μg/mL | pspA-DT(Xba1, Pst1) 26.1μg/mL | J23107-tatABCD(EcoR1, Spe1) 1.5μg/mL | Ligation High Ver.2 | total |
---|---|---|---|---|
1 | 3 | 6 | 5 | 15 |
pSB3C5(EcoR1, Pst1) 26.8μg/mL | MilliQ | Ligation High Ver.2 | total |
---|---|---|---|
1 | 9 | 5 | 15 |
LacP(pSB3C5)(Spe1, Pst1) 10μg/mL | GFP-DT(Xba1, Pst1) 7.0μg/mL | Ligation High Ver.2 | total |
---|---|---|---|
1 | 5 | 3 | 9 |
LacP(pSB3C5)(Spe1, Pst1) 10μg/mL | MilliQ | Ligation High Ver.2 | total |
---|---|---|---|
1 | 5 | 3 | 9 |
4℃, overnight
Observation through a confocal microscope
left:observation with Hoechst
right:observation GFP
September 13
Miniprep
- J23107-tatABCD-pspA-DT(pSB3C5)-1 130μg/mL 1.37(260/280) 0.79(260/230)
Restrictive Digestion
J23107-tatABCD-pspA-DT(pSB3C5) (130μg/mL) | Spe1 | Pst1 | BufferH | MilliQ | total
|
---|---|---|---|---|---|
10 | 1 | 1 | 3 | 15 | 30 |
Transformation
- LacP-GFP generater
- LacP
- J23107-tatABCD-pspA-DT(pSB3C5)
- J23107-tatABCD
Colony PCR
J23107-tatABCD-pspA-DT
→See "September 14 -Colony PCR-"
September 14
Colony PCR
See "September 13 -Colony PCR-"
J23107-tatABCD-pspA-DT
Colony PCR
- J23107-tatABCD-pspA-DT (1-6)
2×Quick Taq | VF | VR | MilliQ | Total |
---|---|---|---|---|
23 | 1 | 1 | 25 | 50 |
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 4min
→30cycles
September 15
Liquid culture
- J23107-tatABCD-pspA-DT-1
- J23107-tatABCD-pspA-DT-2
- J23107-tatABCD-pspA-DT-3
Miniprep
- J23107-tatABCD-pspA-DT-1 22.9μg/mL
- J23107-tatABCD-pspA-DT-2 51.8μg/mL 1.57(260/280) 1.01(260/230)
- J23107-tatABCD-pspA-DT-3 28.5μg/mL 1.57(260/280) 0.94(260/230)
Restrictive Digestion
J23107-tatABCD-pspA-DT (51.8μg/mL) | EcoR1 | Spe1 | BufferM | MilliQ | total |
---|---|---|---|---|---|
15 | 0.5 | 0.5 | 3 | 11 | 30 |
LacP-torA-GFP-DT (79.6μg/mL) | EcoR1 | Xba1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
LacP-kil-DT (56.0μg/mL) | EcoR1 | Xba1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
15 | 0.5 | 0.5 | 3 | 0.5 | 10.5 | 30 |
J23107-tatABCD-pspA-DT (51.8μg/mL) | EcoR1 | Pst1 | BufferM | MilliQ | total |
---|---|---|---|---|---|
15 | 0.5 | 0.5 | 3 | 11 | 30 |
37℃, 2 hours
Electrophoresis
①. 1kb ladder
②. J23107-tatABCD-pspA-DT
③. J23107-tatABCD-pspA-DT(EcoR1, Spe1)
④. J23107-tatABCD-pspA-DT(EcoR1, Pst1)
⑤. LacP-kil-DT
⑥. LacP-kil-DT(EcoR1, Xba1)
⑦. LacP-torA-GFP-DT
⑧. LacP-torA-GFP-DT(EcoR1, Xba1)
Gel extraction
①. 1kb ladder
②. J23107-tatABCD-pspA-DT(EcoR1, Spe1)
③. J23107-tatABCD-pspA-DT(EcoR1, Spe1)
④. J23107-tatABCD-pspA-DT(EcoR1, Pst1)
⑤. J23107-tatABCD-pspA-DT(EcoR1, Pst1)
⑥. 1kb ladder
- J23107-tatABCD-pspA-DT(EcoR1, Spe1) 14.2μg/mL 1.28(260/280) 0.54(260/230)
- J23107-tatABCD-pspA-DT(EcoR1, Pst1) 7.3μg/mL 1.55(260/280) 0.38(260/230)
Purification
- LacP-torA-GFP-DT(EcoR1, Xba1) 7.7μg/mL 1.41(260/280) 0.99(260/230)
- LacP-kil-DT(EcoR1, Xba1) →failed
Ligation
LacP-torA-GFP-DT(EcoR1, Xba1) | J23107-tatABCD-pspA-DT(EcoR1, Spe1) | Ligation High Ver.2 | total |
---|---|---|---|
4 | 10 | 7 | 21 |
pSB1C3(EcoR1, Pst1) | J23107-tatABCD-pspA-DT(EcoR1, Pst1) | Ligation High Ver.2 | total |
---|---|---|---|
4 | 10 | 7 | 21 |
at 16℃ for overnight
September 16
Transformation
LacP-torA-GFP-DT-J23107-tatABCD-pspA-DT
J23107-tatABCD-pspA-DT(pSB1C3)
September 17
Colony PCR
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-6)
J23107-tatABCD-pspA-DT(pSB1C3) (1-6)
J23107-kil-DT (6-10)
Restrictive Digestion
LacP-torA-GFP-DT (126μg/mL) | EcoR1 | Xba1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 1 | 1 | 3 | 3 | 12 | 30 |
LacP-torA-GFP-DT (126μg/mL) | Xba1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|
3 | 1 | 3 | 3 | 20 | 30 |
Electrophoresis
A1-A6. J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-6)
B1-B6. J23107-tatABCD-pspA-DT(pSB1C3) (1-6)
Liquid culture
- J23107-tatABCD-pspA-DT(pSB1C3)
- J23107-tatABCD-pspA-DT(pSB3C5)
- LacP-torA-GFP-DT(pSB3C5)
Electrophoresis
lane1. 1kb ladder
lane2. J23101-kil-DT-6
lane3. J23101-kil-DT-7
lane4. J23101-kil-DT-8
lane5. J23101-kil-DT-10
lane6. LacP-torA-GFP-DT
lane7. LacP-torA-GFP-DT(EcoR1, Xba1)
lane8. LacP-torA-GFP-DT(Xba1)
Miniprep
- J23106-kil-DT 11.7μg/mL 1.93(260/280) 1.46(260/230)
September 18
Gel extraction
- LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL 1.37(260/280) 0.82(260/230)
Ligation
J23107-tatABCD-pspA-DT(EcoR1, Spe1) 14.2μg/mL | LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL | Ligation High Ver.2 | total |
---|---|---|---|
8 | 1.5 | 9.5 | 19 |
LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL | Ligation High Ver.2 | total |
---|---|---|
1.5 | 1.5 | 3 |
at 4℃ for 1 hour
Transformation
- J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT
- LacP-torA-GFP-DT(EcoR1, Xba1)
Observation through a confocal microscope
We made sample for observation through a confocal microscope.
We incubated sample with 0mM/0.5mM IPTG for 1.5 hours.
Then, we incubated onto medium without IPTG for 2.5 hours.
IPTG(mM) | medium |
---|---|
0 | LB |
0.1 | LB |
0.1 | LB with 0.1mM IPTG (positive control) |
0 | LB with 2 percent glucose |
0.1 | LB with 2 percent glucose |
0 | LB with 5 percent glucose |
0.1 | LB with 5 percent glucose |
0 | LB with 2 percent glucose (make medium new after incubate for 1.5 hours) |
0 | LB with 2 percent glucose (make medium new after incubate for 1.5 hours) |
Restrictive Digestion
pSB4K5 | EcoR1 | Pst1 | BufferH | MilliQ | total |
---|---|---|---|---|---|
10 | 1 | 1 | 3 | 15 | 30 |
pSB4K5 | EcoR1 | BufferH | MilliQ | total |
---|---|---|---|---|
3 | 0.5 | 1 | 5.5 | 30 |
pSB4K5 | Pst1 | BufferH | MilliQ | total |
---|---|---|---|---|
3 | 0.5 | 1 | 5.5 | 10 |
LacP-torA-GFP-DT | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 1 | 1 | 3 | 3 | 12 | 30 |
LacP-torA-GFP-DT | Xba1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|
2 | 1 | 1 | 1 | 5 | 10 |
LacP-torA-GFP-DT | Pst1 | BufferH | MilliQ | total |
---|---|---|---|---|
2 | 1 | 1 | 6 | 10 |
37℃, 2 hours
Gel extraction
- LacP-torA-GFP-DT(Xba1, Pst1) 9.7μg/mL 1.34(260/280) 0.62(260/230)
- pSB4K5(EcoR1, Pst1) 9.6μg/mL 1.28(260/280) 0.32(260/230)
Miniprep
- J23107-tatABCD-psp-DT(pSB1C3) 78μg/mL
- J23107-tatABCD-psp-DT(pSB1C3) 48μg/mL
- LacP-torA-GFP-DT 29μg/mL
- J23101-kil-DT 25μg/mL
Restrictive Digestion
J23101-Kil-DT 25µg/ml | BufferM | Xba1 | Pst1 | BSA | MilliQ | total |
---|---|---|---|---|---|---|
40 | 6 | 1 | 1 | 6 | 6 | 60 |
J23101-Kil-DT 25µg/ml | BufferM | Xba1 | BSA | MilliQ | total |
---|---|---|---|---|---|
3 | 1 | 0.5 | 1 | 4.5 | 10 |
J23101-Kil-DT 25µg/ml | BufferH | Pst1 | MilliQ | total |
---|---|---|---|---|
3 | 1 | 0.5 | 5.5 | 60 |
at 37℃ for 2 hours
Electrophoresis
①. pSB4K5
②. pSB4K5(Pst1)
③. pSB4K5(EcoR1)
④. pSB4K5(EcoR1, Pst1)
⑤. LacP-torA-GFP-DT
⑥. LacP-torA-GFP-DT(Xba1)
⑦. LacP-torA-GFP-DT(Pst1)
⑧. LacP-torA-GFP-DT(Xba1, Pst1)
①. 1kb ladder
②. J23101-Kil-DT(Xba1, Pst1)
③. J23101-Kil-DT(Xba1, Pst1)
④. J23101-Kil-DT(Xba1, Pst1)
⑤. J23101-Kil-DT(Xba1, Pst1)
⑥. 1kb ladder
Restrictive Digestion
J23101-Kil-DT | BufferM | Xba1 | Pst1 | BSA | MilliQ | total |
---|---|---|---|---|---|---|
15 | 3 | 1 | 1 | 3 | 7 | 30 |
J23101-Kil-DT | BufferM | Xba1 | BSA | MilliQ | total |
---|---|---|---|---|---|
3 | 1 | 0.5 | 1 | 4.5 | 10 |
J23101-Kil-DT | BufferH | Pst1 | MilliQ | total |
---|---|---|---|---|
3 | 1 | 0.5 | 5.5 | 10 |
37℃
September 19
Gel extraction
lane1. 1kb ladder
lane2. pSB4K5(EcoR1, Pst1)
lane3. pSB4K5(EcoR1, Pst1)
lane4. empty
lane5. LacP-torA-GFP-DT(Xba1, Pst1)
lane6. LacP-torA-GFP-DT(Xba1, Pst1)
Ligation
LacP-torA-GFP-DT(Xba1,Pst1) 9.7μg/mL | pSB4K5(EcoR1,Pst1) 9.6μg/mL | J23107-tatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mL | Ligation High Ver.2 | total |
---|---|---|---|---|
7 | 2 | 7 | 8 | 24μL |
pSB4K5(EcoR1,Pst1) 9.6μg/mL | Ligation High Ver.2 | MilliQ | total |
---|---|---|---|
2 | 1 | 21 | 24μL |
incubate at 37℃ for 2h
Electrophoresis
J23101-kil-DT (Xba1,Pst1), (Xba1), (Pst1)
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: (Xba1)
Lane5: (Pst1)
Lane6: (Xba1, Pst1)
Liquid Culture
We did liquid culture J23106-kil-DT(9/10, Amp)'s 6,7 at LB mediumAmp.
Transformation
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT
pSB4K5 negative control
Liquid culture
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:1~5
Colony PCR
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT
VF2 | VR | Quick tag | MilliQ | total |
---|---|---|---|---|
1 | 1 | 25 | 23 | 50 |
Predenature 94℃ 2min
Denature 94℃ 30sec
Annealing 55℃ 30sec
Extension 68℃ 5min
→30cycles
Restrictive Digestion
LacP-kil-DT(4) 62.5μg/mL | Xba1 | Pst1 | 10xBSA | MilliQ | BufferM | total |
---|---|---|---|---|---|---|
10 | 1 | 1 | 3 | 12 | 3 | 30 |
LacP-kil-DT(4) | EcoR1 | Pst1 | MilliQ | BufferH | total |
---|---|---|---|---|---|
10 | 1 | 1 | 15 | 3 | 30 |
LacP-kil-DT | Xba1 | 10xBSA | MilliQ | BufferM | total |
---|---|---|---|---|---|
2 | 0.5 | 1 | 5.5 | 1 | 10 |
LacP-kil-DT | EcoR1 | MilliQ | BufferH | total |
---|---|---|---|---|
2 | 0.5 | 6.5 | 1 | 10 |
LacP-kil-DT | Pst1 | MilliQ | BufferH | total |
---|---|---|---|---|
2 | 0.5 | 6.5 | 1 | 10 |
at 37℃, 2 hours
Electrophoresis
LacP-kil-DT
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: (EcoR1)
Lane5: (Xba1)
Lane6: (Pst1)
Lane7: (EcoR1, Pst1)
Lane8: (Xba1, Pst1)
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(1-5)
Lane1: ladder
Lane2: 1
Lane3: 2
Lane4: 3
Lane5: 4
Lane6: 5
Ligation
LacP-torA-GFP-DT(Xba1,Pst1) 9.7μg/mL | pSB4K5(EcoR1,Pst1) 9.6μg/mL | J23107-tatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mL | Ligation High Ver.2 | total |
---|---|---|---|---|
7 | 4 | 7 | 9 | 27μL |
(Negative control)
pSB4K5(EcoR1,Pst1) 9.6μg/mL | Ligation High Ver.2 | MilliQ | total |
---|---|---|---|
4 | 9 | 14 | 27μL |
Restrictive Digestion
torA-R9 101.8μg/mL | bufferH | MilliQ | EcoR1 | Pst1 | total |
---|---|---|---|---|---|
10 | 3 | 15 | 1 | 1 | 30 |
torA-R9 101.8μg/mL | bufferH | MilliQ | EcoR1 | Pst1 | total |
---|---|---|---|---|---|
3 | 1 | 5.5 | 0.5 | 0 | 10 |
torA-R9 101.8μg/mL | bufferH | MilliQ | EcoR1 | Pst1 | total |
---|---|---|---|---|---|
3 | 1 | 5.5 | 0 | 0.5 | 10 |
PCR
torA-R9 | MilliQ | 10xPCR buffer for KOD plus neo | 2mM dNTP3 | 25mM MgSO4 | M13 | KOD plus neo | total |
---|---|---|---|---|---|---|---|
0.5 | 34 | 5 | 5 | 3 | 1.5 | 1 | 50 |
Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 60℃ 30sec
Extension 68℃ 20sec
→35cycles
Electrophoresis
torA-R9 plasmid, (EcoR1), (Pst1), (EcoR1,Pst1)
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: EcoR1
Lane5: Pst1
Lane6: EcoR1,Pst1
Lane7: empty
Lane8: 1kb ladder
Liquid culture
LacP-tatA-GFP-DT (9/4)
PCR purification
torA-R9 68.0μg/mL
September 20
Gel extraction
LacP-kil-DT (EcoR1,Pst1) (Xba1,Pst1)
Restrictive Digestion
torA-R9 68.0μg/mL | bufferM | Spe1 | MilliQ | Total |
---|---|---|---|---|
20 | 3 | 1 | 6 | 30 |
at 37℃, 2h
Ligation
LacP-kil-DT(Xba1,Pst1) 12.4μg/mL | pSB1C3(EcoR1,Pst1) 34.2μg/mL | J23107-tatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mL | Ligation High Ver.2 | total |
---|---|---|---|---|
7 | 4 | 7 | 9 | 27μL |
(Negative control)
pSB1C3(EcoR1,Pst1) | Ligation High Ver.2 | MilliQ | total |
---|---|---|---|
4 | 9 | 14 | 27μL |
Transformation
Using above Ligation and Negative control's product and torA-R9.
Miniprep
J23106-kil-DT 6; 12.0μg/mL, (260/280): 1.78, (260/230): 1.73
J23106-kil-DT 7; 13.6μg/mL, (260/280): 1.85, (260/230): 1.73
Restrictive Digestion
GFP-DT 84.1μg/mL | bufferM | 10xBSA | BbaI | MilliQ | total |
---|---|---|---|---|---|
15 | 3 | 3 | 1 | 8 | 30 |
at 37℃, 2 hours
Colony PCR
We made J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT's 1~8 samples.
Quick tag | tat f primer | VR | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
Predenature 94℃ 2min
Denature 94℃ 30sec
Annealing 54℃ 30sec
Extension 68℃ 4min
→30cycles
J23701-tatABCD-pspA-DT-LacP-torA-GFP-DT
Lane1: 1kb ladder
Lane2: 1
Lane3: 2
Lane4: 3
Lane5: 4
Lane6: 5
Lane7: 6
Lane8: 7
Lane9: 8
Lane10: 1kb ladder
Lane11: empty
Lane12: empty
→Bands didn't appear so we do PCR for 10 more cycles.
Liquid culture
We did liquid culture J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT's 1~4 samples.
Gel extraction
torA-R9 (EcoR1,Pst1): 31μg/mL, (280/280): 0.80, (280/230): 0.39
pSB1C3(EcoR1,Pst1): 22μg/mL, (280/280):1.33, (280/230):0.6
torA-R9 (Spe1): 56μg/mL, (280/280): 1.11, (280/230): 0.69
GFP-DT(Xba1): 45μg/mL, (280/280): 1.15, (280/230): 0.88
Ligation
torA-R9 (EcoR1,Pst1) | pSB1C3(EcoR1,Pst1) | Ligation High Ver.2 | total |
---|---|---|---|
8 | 2 | 5 | 15μL |
torA-R9 (Spe1) | pSB1C3(Xba1) | Ligation High Ver.2 | total |
---|---|---|---|
3 | 3 | 3 | 9μL |
at 16℃, 1hour
Colony PCR
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (4k5)
numbering 9~15
Transformation
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (4k5)
J23107-tatABCD-pspA-DT-LacP-torA-kil-DT(pSB1C3)
Negative control (pSB1C3)
Negative control(pSB4K5)
torA-R9(pSB1C3)
Liquid culture
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT: colony PCR(9~15)
2xQuick tag | VF | VR | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
Predenature 94℃ 2min
Denature 94℃ 30sec
Annealing 55℃ 30sec
Extension 68℃ 5min
→30cycles
PCR
torA-R9-GFP-DT | MilliQ | KOD plus ver.2 buffer | dNTP3 | MgSO4 | M13 | VR | KOD plus | total |
---|---|---|---|---|---|---|---|---|
1 | 35 | 5 | 5 | 2 | 1.5 | 1.5 | 1 | 52 |
Predenature 94℃ 2min
Denature 94℃ 15sec
Annealing 55℃ 30sec
Extension 68℃ 5min
→30cycles
Miniprep
J23701-tatABCD-pspA-DT-LacP-torA-GFP-DT:2,3
2: 10.4μg/mL, (250/280): 1.45, (260/230): 0.95
3: 6.0μg/mL, (250/280): 1.32, (260/230): 0.79
(*)100μL
Electrophoresis
torA-R9-GFP-DT(after PCR)
September 21
Gel extraction
torA-R9-GFP-DT(after PCR): 10.3μg/mL, (260/280): 1.28, (220/230): 0.49
Electrophoresis
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(kan): 9~15
Electrophoresis
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT: 1,2
Lane1: empty
Lane2: empty
Lane3: 2
Lane4: 3
Lane5: 1kb ladder
Restrictive Digestion
LacP-kil-DT(3) | Xba1 | Pst1 | 10xBSA | MilliQ | BufferM | total |
---|---|---|---|---|---|---|
15 | 1 | 1 | 3 | 7 | 3 | 30 |
LacP-torA-GFP-DP(9/5) | Xba1 | Pst1 | 10xBSA | MilliQ | BufferM | total |
---|---|---|---|---|---|---|
15 | 1 | 1 | 3 | 7 | 3 | 30 |
LacP-kil-DT | Xba1 | 10xBSA | MilliQ | BufferM | total |
---|---|---|---|---|---|
2 | 0.5 | 1 | 5.5 | 1 | 10 |
LacP-torA-GFP-DP | Xba1 | 10xBSA | MilliQ | BufferM | total |
---|---|---|---|---|---|
2 | 0.5 | 1 | 5.5 | 1 | 10 |
LacP-kil-DT | Pst1 | MilliQ | BufferH | total |
---|---|---|---|---|
2 | 0.5 | 6.5 | 1 | 10 |
LacP-torA-GFP-DP | Xba1 | Pst1 | MilliQ | BufferH | total |
---|---|---|---|---|---|
2 | 0.5 | 1 | 6.5 | 1 | 10 |
J23107-tatABCD-pspA-DT | Spe1 | Pst1 | BufferH | MilliQ | total |
---|---|---|---|---|---|
20 | 1 | 1 | 4 | 14 | 40 |
J23107-tatABCD-pspA-DT | Spe1 | BufferM | MilliQ | total |
---|---|---|---|---|
2 | 0.5 | 1 | 6.5 | 10 |
J23107-tatABCD-pspA-DT | Pst1 | BufferH | MilliQ | total |
---|---|---|---|---|
2 | 0.5 | 1 | 6.5 | 10 |
at 37℃, 2hours
Electrophoresis
lane1. 1kb ladder
lane2. empty
lane3. LacP-kil-DT plasmid
lane4. LacP-kil-DT(Xba1)
lane5. LacP-kil-DT(Pst1)
lane6. LacP-kil-DT(Xba1, Pst1)
lane7. empty
lane8. LacP-torA-GFP-DP plasmid
lane9. LacP-torA-GFP-DP(Xba1)
lane10. LacP-torA-GFP-DP(Pst1)
lane11. LacP-torA-GFP-DP(Xba1, Pst1)
lane12. 1kb ladder
lane1. 1kb ladder
lane2. empty
lane3. J23107-tatABCD-pspA-DT plasmid
lane4. J23107-tatABCD-pspA-DT(Spe1)
lane5. J23107-tatABCD-pspA-DT(Pst1)
lane6. J23107-tatABCD-pspA-DT(Spe1, Pst1)
Colony PCR
- torA-R9(pSB1C3) (1-4)
10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | Total |
---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→30cycles
Electrophoresis
lane1. 1kb ladder
lane2. empty
lane3. torA-R9-1
lane4. torA-R9-2
lane5. torA-R9-3
lane6. torA-R9-4
lane7. empty
lane8. empty
September 22
Miniprep
torA-R9(Amp)1: 113.7μg/mL, (260/280): 1.96, (280/230): 2.42
torA-R9(Amp)2: 97.8μg/mL, (260/280): 1.67, (280/230): 1.72
torA-R9(Amp)3: 104.2μg/mL, (260/280): 1.93, (280/230): 2.67
LacP-torA-GFP-DT 6: 37.4μg/mL, (260/280): 2.24, (280/230): 3.27
Electrophoresis
LaneA: J23107-tatABCD (9/6)
LaneB: J23107-tatABCD-pspA-DT pSB3C5 (9/13)
LaneC: J23107-tatABCD-pspA-DT 1C5 (9/15)
LaneD: J23107-tatABCD-pspA-DT (9/15)
LaneE: J23107-tatABCD-pspA-DT(pSB1C3) (9/18)
LaneF: J23107-tatABCD-pspA-DT(pSB3C5) (9/18)
LaneG: J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:2 (9/20)
LaneH: J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:3 (9/20)
LaneA~H are (EcoR1,Pst1)
Restrictive Digestion
J23107-tatABCD-pspA-DT-LacPtorA-GFP-DT:2 | BufferH | Pst1 | MilliQ | total |
---|---|---|---|---|
15 | 3 | 1 | 12 | 30 |
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:3 | BufferH | Pst1 | MilliQ | total |
---|---|---|---|---|
15 | 3 | 1 | 12 | 30 |
Restrictive Digestion
LacP-torA-GFP-DT(37.4μg/mL) | BufferH | EcoR1 | Pst1 | total |
---|---|---|---|---|
25 | 3 | 1 | 1 | 30 |
Electrophoresis
①: J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:2
②: LacP-torA-GFP-DT(EcoR1,Pst1) (9/22)
③: J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:3
Gel extraction
LacP-torA-GFP-DT(EcoR1,Pst1) 5.5μg/ml 1.21(260/280) 0.37(260/230)
Purification
LacP-torA-GFP-DT 78.4μg/ml 1.62(260/280) 1.03(260/230)
Restrictive Digestion
J23107-tatABCD-pspA-DT(pSB1C3) 78μg/ml | BufferH | Spe1 | Pst1 | MilliQ | total |
---|---|---|---|---|---|
15 | 3 | 1 | 1 | 10 | 30 |
J23107-tatABCD-pspA-DT(pSB1C3) 78μg/ml | BufferH | Spe1 | MilliQ | total |
---|---|---|---|---|
3 | 1 | 0.5 | 5.5 | 10 |
J23107-tatABCD-pspA-DT(pSB1C3) 78μg/ml | BufferH | Pst1 | MilliQ | total |
---|---|---|---|---|
3 | 1 | 0.5 | 5.5 | 10 |
LacP-torA-GFP-DT 78.4μg/ml | BufferM | BSA | Xba1 | Pst1 | MilliQ | total |
---|---|---|---|---|---|---|
15 | 3 | 3 | 1 | 1 | 7 | 30 |
LacP-torA-GFP-DT 78.4μg/ml | BufferM | BSA | Xba1 | MilliQ | total |
---|---|---|---|---|---|
3 | 1 | 1 | 0.5 | 4.5 | 10 |
LacP-torA-GFP-DT 78.4μg/ml | BufferH | Pst1 | MilliQ | total |
---|---|---|---|---|
3 | 1 | 0.5 | 5.5 | 10 |
at 37℃ for 2 hours
Electrophoresis
Lane1: 1kb ladder
Lane2: empty
Lane3: J23107-tatABCD-pspA-DT(pSB1C3)
Lane4: J23107-tatABCD-pspA-DT(Spe1, Pst1)
Lane5: J23107-tatABCD-pspA-DT(Spe1)
Lane6: J23107-tatABCD-pspA-DT(Pst1) (9/18)
Lane7: empty
Lane8: LacP-torA-GFP-DT
Lane9: LacP-torA-GFP-DT(Xba1, Pst1)
Lane10: LacP-torA-GFP-DT(Xba1)
Lane11: LacP-torA-GFP-DT(Pst1)
Lane12: empty
Lane13: 1kb ladder
September 23
Gel extraction
- LacP-torA-GFP-DT(EcoR1, Pst1)
lane1 1kb ladder
lane3 DNA
lane4 DNA
lane5 DNA
→failed
lane1 1kb ladder
lane3 J23107-tatABCD-pspA-DT(pSB1C3)(Spe1, Pst1)
lane5 LacP-torA-GFP-DT(Xba1, Pst1)
→failed
Restrictive Digestion
LacP-torA-GFP-DT (78.4μg/mL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
15 | 1 | 1 | 3 | 3 | 7 | 30 |
LacP-torA-GFP-DT (78.4μg/mL) | Xba1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|
3 | 0.5 | 1 | 1 | 4.5 | 10 |
J23107-tatABCD-pspA-DT(pSB1C3) (78μg/mL) | Spe1 | Pst1 | BufferH | MilliQ | total |
---|---|---|---|---|---|
9 | 1 | 1 | 2 | 7 | 20 |
Ligation
LacP-torA-GFP-DT(EcoR1, Pst1) (5.5μg/mL) | pSB4K5 (9.6μg/mL) | Ligation High Ver.2 | total |
---|---|---|---|
8 | 4 | 6 | 18 |
at 16℃ for 1 hour
Liquid culture
- LacP-tatABCD-pspA-DT (2, 3, 5, 6)
Colony PCR
LacP-tatABCD-pspA-DT (1-6)
2×Quick Taq | VF2 | VR | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 4min
→30cycles
Electrophoresis
①, LacP-torA-GFP-DT
②, LacP-torA-GFP-DT(Xba1, Pst1)
③, LacP-torA-GFP-DT(Xba1)
④, LacP-torA-GFP-DT(Pst1)
⑤, J23107-tatABCD-pspA-DT(Spe1, Pst1)
Liquid culture
- LacP-torA-GFP-DT (4, 7)
- LacP-GFP generater (1-3)
- T7-6His-GFP-DT (1, 2)
Gel extraction
- LacP-torA-GFP-DT (Xba1, Pst1) 6.8μg/mL 1.27(260/280) 0.40(260/230)
Purification
- J23107-tatABCD-pspA-DT(Spe1, Pst1) 15.1μg/mL 1.49(260/280) 0.80(260/230)
Ligation
J23107-tatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL) | LacP-torA-GFP-DT (Xba1, Pst1) (6.8μg/mL) | Ligation High Ver.2 | total |
---|---|---|---|
10 | 4 | 7 | 21 |
J23107-tatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL) | LacP-kil-DT (Xba1, Pst1) (12.4μg/mL) | Ligation High Ver.2 | total |
---|---|---|---|
8 | 2 | 5 | 15 |
at 16℃ for 1 hour
Transformation
LacP-torA-GFP-DT(pSB4K5)
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT
J23107-tatABCD-pspA-DT-LacP-kil-DT
DNA | competent cell | total |
---|---|---|
2 | 10 | 12 |
preculture at 37℃ for 1 hour culture at 37℃ for 16 hours
Electrophoresis
J23107-tatABCD-pspA-DT(pSB1C3) (1-6)
lane1 1kb ladder
lane2~7 DNA
lane8 1kb ladder
September 24
Miniprep
- J23107-tatABCD-pspA-DT-2 139.4μg/ml 1.69(260/280) 1.83(260/230)
- J23107-tatABCD-pspA-DT-3 134.7μg/ml 1.61(260/280) 1.69(260/230)
- J23107-tatABCD-pspA-DT-5 139.3μg/ml 1.55(260/280) 1.45(260/230)
- J23107-tatABCD-pspA-DT-6 138.5μg/ml 1.58(260/280) 1.40(260/230)
- LacP-torA-GFP-DT-4
- LacP-torA-GFP-DT-7
- T7-6His-GFP-DT-1
- T7-6His-GFP-DT-2
Colony PCR
LacP-GFP generater (1-3)
2×Quick Taq | VF2 | VR | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1min
→30cycles
①-③, LacP-GFP generater (1-3)
Liquid culture
- J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-4)
- J23107-tatABCD-pspA-DT-LacP-kil-DT (1-3)
Colony PCR
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-4)
J23107-tatABCD-pspA-DT-LacP-kil-DT (1-3)
2×Quick Taq | VF2 | VR | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 5min
→30cycles
Electrophoresis
lane1, 1kb ladder
lane2-5, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (1-4)
lane6-8, J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (1-3)
September 25
Liquid culture
- J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB4K5)-2
Miniprep
J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3)-3: 26.2μg/mL, (260/280): 1.50, (280/230): 0.65
J23107-tatABCD-pspA-DT-LacP-torA-GFP(pSB1C3)-3: 24.8μg/mL, (260/280): 1.56, (280/230): 0.76
LacP-GFP generater-1: 42.0μg/mL, (260/280): 1.58, (280/230): 0.94
Restrictive Digestion
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (26.2ng/µL) | EcoRl | Spel | buffer M | MiliQ | Total |
---|---|---|---|---|---|
15 | 1 | 1 | 3 | 10 | 30 |
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (26.2ng/µL) | EcoRl | buffer M | MiliQ | Total |
---|---|---|---|---|
2 | 0.5 | 1 | 6.5 | 10 |
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (26.2ng/µL) | Spel | buffer M | MiliQ | Total |
---|---|---|---|---|
2 | 0.5 | 1 | 6.5 | 10 |
J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (24.8ng/µL) | EcoRl | Pstl | buffer H | MiliQ | Total |
---|---|---|---|---|---|
15 | 1 | 1 | 3 | 10 | 30 |
J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (24.8ng/µL) | Pstl | buffer H | MiliQ | Total |
---|---|---|---|---|
2 | 0.5 | 1 | 6.5 | 10 |
J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (24.8ng/µL) | EcoRl | buffer H | MiliQ | Total |
---|---|---|---|---|
2 | 0.5 | 1 | 6.5 | 10 |
at 37℃ for 2 hours
Electrophoresis
lane1, 1kb ladder
lane2, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3)
lane3, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl, Spel)
lane4, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl)
lane5, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (Spel)
lane6, J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3)
lane7, J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (EcoRl, Pstl)
lane8, J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (EcoRl)
lane9, J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (Pstl)
lane10, LacP-torA-GFP-DT(EcoR1,Xba1)(9/15)
lane11, LacP-torA-GFP-DT(EcoR1,Xba1)(9/17)
lane12, 1kb ladder
→The bands didn't appear, so we did one more electrophoresis.
lane1, 1kb ladder
lane2, LacP-torA-GFP-DT(EcoR1,Xba1)(9/17)
lane3, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3)
lane4, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl, Spel)
lane5, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl)
lane6, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (Spel)
Liquid culture
- LacP-torA-GFP-DT(pSB4K5) (1-4)
Restrictive Digestion
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) | EcoRl | Spel | buffer M | MiliQ | Total |
---|---|---|---|---|---|
15 | 1 | 1 | 3 | 10 | 30 |
J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) | EcoRl | Pstl | buffer H | MiliQ | Total |
---|---|---|---|---|---|
15 | 1 | 1 | 3 | 10 | 30 |
at 37℃ for 1 hour
Electrophoresis
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl, Spel)
J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (EcoRl, Pstl)
→The bands didn't appear.
September 6
PCR and Electrophoresis assay
①psB1K3 ②lacI ③GFP ④GFP ⑤RFP ⑥RFP ⑦CFP ⑧DT
Quick Taq | primer F | Primer R | DNA(①-⑧) | milliQ | total |
---|---|---|---|---|---|
12.5 | 0.5 | 0.5 | 1 | 10.5 | 25 |
94℃ 2min, (94℃ 30sec, 50℃ 30sec)x25cycles, 68℃ 50sec
September 7
PCR and Electrophoresis assay of psB1K3
10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(psB1K3) | KOD plus | milliQ | total |
---|---|---|---|---|---|---|---|---|
2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |
94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min
September 10
PCR and Electrophoresis assay of CFP
10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(CFP) | KOD plus | milliQ | total |
---|---|---|---|---|---|---|---|---|
2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |
94℃ 2min, (94℃ 15sec, 48℃ 30sec)x30cycles, 68℃ 45sec
PCR and Electrophoresis assay of lacI
10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(lacI) | KOD plus | milliQ | total |
---|---|---|---|---|---|---|---|---|
2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |
94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 30sec
PCR
①PCR product of psB1K3 ②psB1K3 ③psB1C3 ④psB1C3 ⑤PCR product of CFP
10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(①-⑤) | KOD plus | milliQ | total |
---|---|---|---|---|---|---|---|---|
2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |
94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min20sec
September 11
PCR and Electrophoresis assay
①DT ②PCR product of DT ③PCR product of CFP ④CFP(2011 plate)
10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(①-④) | KOD plus | milliQ | total |
---|---|---|---|---|---|---|---|---|
2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |
94℃ 2min, (94℃ 15sec, 48℃ 30sec)x30cycles, 68℃ 1min
PCR and Electrophoresis assay
①DT 92.1ng/µl ②DT 28.0ng/µl ③DT
10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(①-③) | KOD plus | milliQ | total |
---|---|---|---|---|---|---|---|---|
2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |
94℃ 2min, (94℃ 15sec, 50℃ 30sec)x30cycles, 68℃ 30sec
September 12
Purification of PCR product
psB1K3(9/10④) 55µg/ml lacI(9/10) 90µg/ml GFP(9/6) 42µg/ml RFP(9/6) 71µg/ml DT(9/11) 81µg/ml GFP 37µg/ml RFP 57µg/ml
Golden Gate Assembly
psB1K3 | lacI | GFP(2'-3) | RFP(3'-5) | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
---|---|---|---|---|---|---|---|---|---|---|
1.8 | 0.1 | 1.0 | 0.6 | 0.1 | 1.5 | 0.15 | 1.0 | 1.0 | 8.15 | 15 |
psB1K3 | lacI | GFP(2'-5) | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
---|---|---|---|---|---|---|---|---|---|
1.8 | 0.1 | 1.0 | 0.1 | 1.5 | 0.15 | 1.0 | 1.0 | 8.75 | 15 |
psB1K3 | lacI | GFP(2'-3) | RFP(3'-5) | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
---|---|---|---|---|---|---|---|---|---|---|
2.0 | 0.2 | 1.0 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 1.0 | 6.95 | 15 |
psB1K3 | lacI | GFP(2'-3) | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
---|---|---|---|---|---|---|---|---|---|
2.0 | 0.2 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 1.0 | 7.95 | 15 |
Transformation
①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT
September 13
Transformation
①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT
September 14
Liquid Culture
①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT
September 15
Miniprep
①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT
①4.0 µg/ml ②17 µg/ml ③15 µg/ml ④8.0 µg/ml
Restriction Enzyme Digestion
①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT
DNA(①-④) | 10x NEB Bufer3 | BSA | EcoR1 | MilliQ | Total |
---|---|---|---|---|---|
9 | 2 | 0.5 | 0.5 | 8 | 20 |
September 17
Electrophoresis assay
①lacI-GFP(2'-3)-RFP(3'-5)-DT
②lacI-GFP(2'-5)-DT
③lacI-GFP(2'-3)-RFP(3'-5)-DT
④lacI-GFP(2'-5)-DT
September 18
Restriction Enzyme Digestion
③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT
DNA(③,④) | 10x H Bufer | BSA | EcoR1 | MilliQ | Total |
---|---|---|---|---|---|
15 | 3 | 0.5 | 0.5 | 11 | 30 |
Colony PCR
①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT
2x Quick Tag Dye Mix | VF primer | VR primer | MiliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
September 19
DpnI Restriction Enzyme Digestion of psB1K3
psB1K3 | DpnI | Total |
---|---|---|
10 | 0.2 | 10.2 |
37℃ 1h incubate
Ligation of psB1K3
psB1K3 | Ligation High | MiliQ | TH Kinase | Total |
---|---|---|---|---|
2 | 5 | 7 | 7 | 15 |
16.0℃ 1.5h
September 20
Transformation of psB1K3
September 21
PCR of psB3C5 and Electrophoresis assay
10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(psB3C5) | KOD plus | milliQ | total |
---|---|---|---|---|---|---|---|---|
2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |
94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min
PCR and Electrophoresis assay
①psB3C5 ②psB1K3
10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(①,②) | KOD plus | milliQ | total |
---|---|---|---|---|---|---|---|---|
5.0 | 5.0 | 2.0 | 1.5 | 1.5 | 1.0 | 1.0 | 33 | 50 |
94℃ 2min, (94℃ 15sec, 58℃ 30sec, 68℃ 2min)x30cycles
PCR of DT and Electrophoresis assay
10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(DT) | KOD plus | milliQ | total |
---|---|---|---|---|---|---|---|---|
5.0 | 5.0 | 2.0 | 1.5 | 1.5 | 1.0 | 1.0 | 33 | 50 |
94℃ 2min, (94℃ 15sec, 50℃ 30sec, 68℃ 30sec)x30cycles
September 22
Colony PCR (Colony plated in September 20)
10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA | KOD plus | milliQ | total |
---|---|---|---|---|---|---|---|---|
5.0 | 5.0 | 2.0 | 1.5 | 1.5 | 1.0 | 1.0 | 33 | 50 |
94℃ 2min, (94℃ 15sec, 58℃ 30sec, 68℃ 2min20sec)x25cycles
PCR of Primers and Electrophoresis assay
①J23106(1'-2) ②J23106(2'-3) ③J23106(3'-4) ④J23106(4'-5) ⑤J23106(3'-5) ⑥J23106(2'-5) ⑦J23106(1'-5) ⑧araC(1'-6) ⑨araC(5'-6)
10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA | KOD plus | milliQ | total |
---|---|---|---|---|---|---|---|---|
2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |
94℃ 2min, (94℃ 15sec, 56℃ 30sec)x30cycles, 68℃ 20sec
①J23106(1'-2) 60µg/ml ②J23106(2'-3) 63µg/ml ③J23106(3'-4) 48µg/ml ④J23106(4'-5) 60µg/ml ⑤J23106(3'-5) 62µg/ml ⑥J23106(2'-5) 40µg/ml ⑦J23106(1'-5) 29µg/ml ⑧araC(1'-6) 60µg/ml ⑨araC(5'-6) 62µg/ml
Ligation
4 promoters
psB3C5 | ①J23106(1'-2) | ②J23106(2'-3) | ③J23106(3'-4) | ④J23106(4'-5) | ⑨araC(5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
2.0 | 2.0 | 2.0 | 2.5 | 2.0 | 2.0 | 3.0 | 3.0 | 1.5 | 0.15 | 1.0 | 1.0 | 7.85 | 30 |
3 promoters
psB3C5 | ①J23106(1'-2) | ②J23106(2'-3) | ⑤J23106(3'-5) | ⑨araC(5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
---|---|---|---|---|---|---|---|---|---|---|---|---|
2.0 | 2.0 | 2.0 | 2.5 | 2.0 | 3.0 | 3.0 | 1.5 | 0.15 | 1.0 | 1.0 | 9.85 | 30 |
2 promoters
psB3C5 | ①J23106(1'-2) | ⑥J23106(2'-5) | ⑨araC(5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
---|---|---|---|---|---|---|---|---|---|---|---|
2.0 | 2.0 | 2.5 | 2.0 | 3.0 | 3.0 | 1.5 | 0.15 | 1.0 | 11.85 | 30 |
1 promoter
psB3C5 | ⑦J23106(1'-5) | ⑨araC(5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
---|---|---|---|---|---|---|---|---|---|---|
2.0 | 2.5 | 2.0 | 3.0 | 3.0 | 1.5 | 0.15 | 1.0 | 1.0 | 13.85 | 30 |
0 promoter
psB3C5 | ⑨araC(5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
---|---|---|---|---|---|---|---|---|---|
2.0 | 2.0 | 3.0 | 3.0 | 1.5 | 0.15 | 1.0 | 1.0 | 16.35 | 30 |
September 23
Transformation of product of ligation(9/22)
September 24
Transformation of product of ligation(9/22)
September 25
Liquid Culture
colony1,2,3 of plate2,3
Ligation
4 promoters
psB3C5 | ①J23106(1'-2) | ②J23106(2'-3) | ③J23106(3'-4) | ④J23106(4'-5) | ⑨araC(5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
2.0 | 0.2 | 0.3 | 0.2 | 0.2 | 0.2 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 1.0 | 7.05 | 15 |
3 promoters
psB3C5 | ①J23106(1'-2) | ②J23106(2'-3) | ⑤J23106(3'-5) | ⑨araC(5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
---|---|---|---|---|---|---|---|---|---|---|---|---|
2.0 | 0.2 | 0.3 | 0.2 | 0.2 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 1.0 | 7.25 | 15 |
2 promoters
psB3C5 | ①J23106(1'-2) | ⑥J23106(2'-5) | ⑨araC(5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
---|---|---|---|---|---|---|---|---|---|---|---|
2.0 | 0.2 | 0.3 | 0.2 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 7.45 | 15 |
1 promoter
psB3C5 | ⑦J23106(1'-5) | ⑨araC(5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
---|---|---|---|---|---|---|---|---|---|---|
2.0 | 0.6 | 0.2 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 1.0 | 7.35 | 15 |
0 promoter
psB3C5 | ⑨araC(5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
---|---|---|---|---|---|---|---|---|---|
2.0 | 0.2 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 1.0 | 7.95 | 15 |
(37℃ 3min, 16℃ 4min)x25cycles,