Team:Kyoto/Secretion/Notebook
From 2012.igem.org
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==September 6== | ==September 6== | ||
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====Miniprep==== | ====Miniprep==== | ||
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*J23107-tatABCD-8 82.4μg/mL 2.06(260/280) 2.95(260/230) | *J23107-tatABCD-8 82.4μg/mL 2.06(260/280) 2.95(260/230) | ||
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*J23107-tatABCD-20 102.9μg/mL 2.04(260/280) 2.94(260/230) | *J23107-tatABCD-20 102.9μg/mL 2.04(260/280) 2.94(260/230) | ||
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====Restriction==== | ====Restriction==== | ||
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{| class="wikitable" | {| class="wikitable" | ||
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!J23107-tatABCD 102.9μg/mL||Spe1||Pst1||BufferH||BSA||MilliQ||total | !J23107-tatABCD 102.9μg/mL||Spe1||Pst1||BufferH||BSA||MilliQ||total | ||
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|10||0.5||0.5||3||0.5||15.5||30 | |10||0.5||0.5||3||0.5||15.5||30 | ||
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at 37℃ for 1h | at 37℃ for 1h | ||
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====Transformation==== | ====Transformation==== | ||
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LacP-pspA-DT(pSB3C5) | LacP-pspA-DT(pSB3C5) | ||
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{|class="wikitable" | {|class="wikitable" | ||
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!DNA||Competent cell||total | !DNA||Competent cell||total | ||
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|2||20||22 | |2||20||22 | ||
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|} | |} | ||
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====Gel extraction==== | ====Gel extraction==== | ||
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写真お願いします。<br> | 写真お願いします。<br> | ||
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J23107-tatABCD(Spe1, Pst1) | J23107-tatABCD(Spe1, Pst1) | ||
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→We failed gel extraction because we mistook steps of experiment. | →We failed gel extraction because we mistook steps of experiment. | ||
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====Electrophoresis==== | ====Electrophoresis==== | ||
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写真お願いします。<br> | 写真お願いします。<br> | ||
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LacP-torA-GFP-DT-pspA-DT<br> | LacP-torA-GFP-DT-pspA-DT<br> | ||
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====Restriction==== | ====Restriction==== | ||
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<small>by Terasaka</small><br> | <small>by Terasaka</small><br> | ||
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{| class="wikitable" | {| class="wikitable" | ||
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!J23107-tatABCD||Spe1||Pst1||BufferH||BSA||MilliQ||total | !J23107-tatABCD||Spe1||Pst1||BufferH||BSA||MilliQ||total | ||
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|10||0.5||0.5||3||0.5||15.5||30 | |10||0.5||0.5||3||0.5||15.5||30 | ||
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|} | |} | ||
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====Gel extraction==== | ====Gel extraction==== | ||
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写真お願いします。<br> | 写真お願いします。<br> | ||
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J23107-tatABCD(Spe1, Pst1) | J23107-tatABCD(Spe1, Pst1) | ||
- | + | →See "September 7 -Gel extraction-" | |
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- | →See "September 7 -Gel extraction-" | + | |
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==September 7== | ==September 7== |
Revision as of 10:54, 25 September 2012
Secretion Notebook
February 7
Preculture
We started preculture at 12:10.
February 8
Culture
We start culturing with 300[mL] of LB medium.
time | OD600 |
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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
February 10
Miniprep
DT2 43.9μg/ml(1.34 260/230 1.74 260/280)
lacp1 17.1μg/ml(1.54 260/280 0.83 260/230)
lacp2 18.0μg/ml(1.58 260/280 0.87 260/230)
transformation
B0040 1.4k PsB1A2 B0034 1.2M pSB1A2(from iGEM parts plate)
Competent cell
We did preculture for overnight. We put 1.5mL of preculture on 150mL of LB culture.
time | OD600 |
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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 |
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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 |
Restriction
Const promoterJ23100
DNA | Spe1 | Pst1 | Buffer2 | BSA | MilliQ | total |
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20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
at 37℃ for overnight
February 15
Making gel
1% Agarose gel
Agarose | TAE |
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1.6g | 160mL |
Electrophoresis
Gel No.1
Restriction product | loading dye |
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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 |
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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 |
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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 |
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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 |
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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
Restriction
J23100 | Spe1 | Pst1 | Buffer2 | BSA | MilliQ | total |
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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 | |
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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 |
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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 | |
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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
Competent cell
We put 3mL of preculture product on yesterday onto 300mL of LB medium
time | OD600 |
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10:30 | start |
12:10 | 0.118 |
13:00 | 0.270 |
13:30 | 0.502 |
Transformation
pSB1AT3-2 | competent cell | MilliQ | total |
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0 | 20 | 10 | 30 |
2 | 20 | 8 | 30 |
10 | 20 | 0 | 30 |
- Results(on Feb. 19)
pSB1AT3-2 | number of colony |
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0 | 0 |
2 | 177 |
10 | 590 |
Transformation efficiency 7.4x10^4 colonys/μg
February 20
Restriction
sample 1
DT plasmid | EcoR1 | Xba1 | Buffer | BSA | MilliQ | total |
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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
lane_1 1kb ladder
lane_2 a
lane_3 b
lane_4 a
lane_5 b
lane_6 a
lane_7 b
lane_8 1kb ladder
- sample2
c. sample2 2μL + MilliQ 3μL + 6×Loading Dye 1μL
d. sample2 5μL + 6×Loading Dye 1μL
lane_1 1kb ladder
lane_2 c
lane_3 d
lane_4 1kb ladder
PCR
Buffer | dNTPs | MgSO4 | primer-f | primer-r | genomic DNA | KOD plus | MilliQ | total | |
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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 | |
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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
lane_1.1kb ladder
lane_2.tatABCD
lane_3.tatABCD
lane_4.TAMO1
lane_5.TAMO2
lane_6.1kb ladder
lane_1.1kb ladder
lane_2.tatABCD1
lane_3.tatABCD2
lane_4.TAMO
lane_5.TAMO
lane_6.1kb 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
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
Restriction
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
Restriction
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
- evernoteに写真なし!至急求む
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
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
Restriction
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
Restriction
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
Restriction
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
Restriction
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
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
Restriction
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
Restriction
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
(31)
Restriction
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
Restriction
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
pspAはコロニーPCR
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
Restriction
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.
Restriction
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 |
Restriction
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
Restriction
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
Restriction
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
Restriction
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 |
Restriction
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
Restriction
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.
Restriction
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
Restriction
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
Restriction
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℃.
Restriction
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→×
Restriction
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
Restriction
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
Restriction
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.
Restriction
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
Restriction
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]
Restriction
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
Restriction Enzyme Processing
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
torAGFP+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
Restriction Enzyme Processing
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
Restriction Enzyme processing
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 |
Electrophoresis
positive control(GFP+DT)
negative control(MilliQ)
① ~⑥ colony PCR lacP+kil+DT
次の写真
① 1kb ladder
② Positive control (GFP+DT)
③ Negative control (MilliQ)
④ ~⑨ lacP+kil+DT ①~⑥
⑩~⑫ pspA 10μL ,6×buffer 2μL
⑬ 1kb ladder
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
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
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
Restriction
J23113(201.0ng/μL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 1 | 1 | 3 | 0.5 | 14.5 | 30 |
Electrophoresis
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
Restriction
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
写真貼ってください
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)
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 |
- negative control for pspA
We did PCR without a template.
- GFP-DT
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
Restriction
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
写真お願いします。
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
写真お願いします。
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-"
Restriction
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
Restriction
pSB1C3(82.9μg/mL) | EcoR1 | Pst1 | BufferH | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
→Gel extraction
Electrophoresis
写真お願いします。
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
写真お願いします。
Purification of PCR products
torA-GFP →See "August 21 -PCR-"
Restriction
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-"
Restriction
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 |
写真お願いします。
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
evernoteに結果のグラフがあります。
Liquid culture
- pspA-pSB1C3(1-3)
- pspA-pSB3C5(1-3)
Restriction
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
写真お願いします。
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
Restriction
pspA-pSB1C3 | EcoR1 | Spe1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
15 | 0.5 | 0.5 | 5 | 0.5 | 28.5 | 50 |
at 37℃ for 1h
Electrophoresis
写真お願いします。
1. 1kb ladder
3. pspA-pSB1C3(EcoR1, Spe1)
August 31
Gel extraction
写真お願いします。
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
Restriction
LacP-torA-GFP-DT | EcoR1 | Spe1 | BufferH | MilliQ | total |
---|---|---|---|---|---|
10 | 1 | 1 | 2 | 6 | 20 |
at 4℃ for overnight
Electrophoresis
写真お願いします。
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
写真お願いします。
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)
写真お願いします。
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
写真お願いします。
LacP-torA-GFP-DT →See "September 5 -Gel extraction-"
Restriction
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
写真お願いします。
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
写真お願いします。
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
写真お願いします。
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
写真お願いします。
1-8. Lacp-torA-GFP-DT
写真お願いします。
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)
Restriction
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
写真お願いします。
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
写真お願いします。
Lacp-torA-GFP-DT(8-15)
写真お願いします。
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)
Restriction
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
写真お願いします。
J23107-tatABCD(Spe1, Pst1)
→We failed gel extraction because we mistook steps of experiment.
Electrophoresis
写真お願いします。
LacP-torA-GFP-DT-pspA-DT
Restriction
by Terasaka
J23107-tatABCD | Spe1 | Pst1 | BufferH | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
Gel extraction
写真お願いします。
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
Restriction
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
写真お願いします。
① -⑧. LacP-pspA-DT
1-8. LacP-torA-GFP-DT
Gel extraction
写真お願いします。
- J23107-tatABCD(EcoR1, Spe1)
- J23107-tatABCD(Pst1, Spe1) ①57.6μg/mL ②23.4μg/mL
- GFP-DT(Xba1, Pst1)
Restriction
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
Restriction
pspA-DT-3(322μg/mL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 1 | 1 | 3 | 3 | 12 | 30 |
at 37℃ for 1h
September 9
Restriction
J23107-tatABCD(102.9μg/mL) | EcoR1 | Spe1 | BufferM | MilliQ | total |
---|---|---|---|---|---|
10 | 1 | 1 | 3 | 15 | 30 |
Electrophoresis
写真お願いします。
1. J23107-tatABCD(EcoR1, Spe1)
2. J23107-tatABCD(before restriction)
3. pspA-DT(Xba1, Pst1)
4. pspA-DT(before restriction)
Gel extraction
写真(2枚)お願いします。
- 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 |
Restriction
写真お願いします。
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
写真(2まい)お願いします。
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
写真お願いします。
- J23107-tatABCD-DT (1-8)
- J23107-tatABCD-pspA-DT (1-8)
Restriction
写真お願いします。
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
写真お願いします。
- 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
写真お願いします。
- 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
写真お願いします。
- J23107-tatABCD-pspA-DT
写真お願いします。
- J23101-kil-DT (1-7)
Restriction
pspA(3C5) (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
写真お願いします。
- pspA(3C5)(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
写真お願いします。
- pspA(3C5)(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
Evernoteに上がっている写真をお願いします。
September 13
Miniprep
- J23107-tatABCD-pspA-DT(pSB3C5)-1 130μg/mL 1.37(260/280) 0.79(260/230)
Restriction
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
写真お願いします。
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)
Restriction
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
写真お願いします。
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
写真お願いします。
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)
Restriction
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
写真お願いします。
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
写真お願いします。
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
写真お願いします。
- 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 |
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 |
0 | LB ここ途中 |
0 | LB |
0 | LB |
Restriction enzyme processing
J23101-Kil-DT | BufferM | Xba1 | Pst | 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 | Pst | MilliQ | total |
---|---|---|---|---|
3 | 1 | 0.5 | 5.5 | 10 |
37℃
September 19
GeneClean II
LacP-torA-GFP-DT
4k5
Ligation
LacP-torA-GFP-DT(X,P) 9.7μg/mL | pSB4K5(E,P) 9.6μg/mL | J23107-tatABCD-PsPA-DT(E,S) 14.2μg/mL | Ligation High Ver.2 | total |
---|---|---|---|---|
7 | 2 | 7 | 8 | 24μL |
pSB4K5(E,P) 9.6μg/mL | Ligation High Ver.2 | MilliQ | total |
---|---|---|---|
2 | 1 | 21 | 24μL |
incubate at 37℃ for 2h
Electrophoresis
J23101-kil-DT(X,P) (X) (P)
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: X
Lane5: P
Lane6: X,P
Liquid Culture
We did liquid culture J23106-kil-DT(9/10, Amp)'s 6,7 at LB mediumAmp.
Transformation
J2-tat-psp-DT-LacP-torAGFP-DT
pSB4K5 negative control
Liquid culture
J2-tat-psp-DT-LacP-torAGFP-DT:1~5
Colony PCR
J2-tat-psp-DT-LacP-torAGFP-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
Restriction enzyme processing
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
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: E
Lane5: X
Lane6: P
Lane7: E,P
Lane8: X,P
Electrophoresis
J23107-tatABCD-pspA-DT-LacP-torAGFP-DT
Lane1: ladder
Lane2: 1
Lane3: 2
Lane4: 3
Lane5: 4
Lane6: 5
Ligation
LacP-torA-GFP-DT(X,P) 9.7μg/mL | pSB4K5(E,P) 9.6μg/mL | J23107-tatABCD-PsPA-DT(E,S) 14.2μg/mL | Ligation High Ver.2 | total |
---|---|---|---|---|
7 | 4 | 7 | 9 | 27μL |
(Negative control)
pSB4K5(E,P) 9.6μg/mL | Ligation High Ver.2 | MilliQ | total |
---|---|---|---|
4 | 9 | 14 | 27μL |
Restriction enzyme processing
torA-R9 101.8μg/mL | bufferH | MilliQ | EcoR1 | Pst | total |
---|---|---|---|---|---|
10 | 3 | 15 | 1 | 1 | 30 |
torA-R9 101.8μg/mL | bufferH | MilliQ | EcoR1 | Pst | total |
---|---|---|---|---|---|
3 | 1 | 5.5 | 0.5 | 0 | 10 |
torA-R9 101.8μg/mL | bufferH | MilliQ | EcoR1 | Pst | 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,(E),(P),(E,P)
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: E
Lane5: P
Lane6: E,P
Lane7: empty
Lane8: 1kb ladder
Liquid culture
LacP-tatA9FT-DT (9/4)
PCR purification
torA-R9 68.0μg/mL
September 20
Gel extraction
LacP-kil-DT (E,P) (X,P)
Restriction enzyme processing
torA-R9 68.0μg/mL | bufferM | Spe1 | MilliQ | Total |
---|---|---|---|---|
20 | 3 | 1 | 6 | 30 |
at 37℃, 2h
Ligation
LacP-kil-DT(X,P) 12.4μg/mL | pSB1C3(E,P) 34.2μg/mL | J23107-tatABCD-PsPA-DT(E,S) 14.2μg/mL | Ligation High Ver.2 | total |
---|---|---|---|---|
7 | 4 | 7 | 9 | 27μL |
(Negative control)
pSB1C3(E,P) | 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
Restriction enzyme processing
GFP-DT 84.1μg/mL | bufferM | 10xBSA | BbaI | MilliQ | total |
---|---|---|---|---|---|
15 | 3 | 3 | 1 | 8 | 30 |
at 37℃, 2 hours
Colony PCR
We made J2-tat-psp-DT-LacP-torAGFP-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
Liquid culture
We did liquid culture J2-tat-psp-DT-LacP-torAGFP-DT's 1~4 samples.
Electrophoresis
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
torA-R9 (E,P): 31μg/mL, (280/280): 0.80, (280/230): 0.39
pSB1C3(E,P): 22μg/mL, (280/280):1.33, (280/230):0.6
torA-R9 (S): 56μg/mL, (280/280): 1.11, (280/230): 0.69
GFP-DT(X): 45μg/mL, (280/280): 1.15, (280/230): 0.88
Ligation
torA-R9 (E,P) | 1C3(E,P) | Ligation High Ver.2 | total |
---|---|---|---|
8 | 2 | 5 | 15μL |
torA-R9 (S) | 1C3(X) | 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-tat-psp-DT-lacP-torA-GFP-DT (4k5)
J23107-tat-psp-DT-lacP-torA-kil-DT(1C3)
Negative control (1C3)
Negative control(4K5)
torA-R9(1C3)
Liquid culture
J23107-tat-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
J2301-tat-psp-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-tat-psp-DT-LacP-torA-GFP-DT(kan): 9~15
Electrophoresis
J2-tat-psp-DT-LacP-torA-GFP-DT: 1,2
Lane1: empty
Lane2: empty
Lane3: 2
Lane4: 3
Lane5: 1kb ladder
Restriction enzyme processing
LacP-kil-DT(3) | Xba1 | Pst1 | 10xBSA | MilliQ | BufferM | total |
---|---|---|---|---|---|---|
15 | 1 | 1 | 3 | 7 | 3 | 30 |
LacP-torA-6FP-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-6FP-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-6FP-DP | Xba1 | Pst1 | MilliQ | BufferH | total |
---|---|---|---|---|---|
2 | 0.5 | 1 | 6.5 | 1 | 10 |
J2-tat-psp-DT | Spe1 | Pst1 | BufferH | MilliQ | total |
---|---|---|---|---|---|
20 | 1 | 1 | 4 | 14 | 40 |
J2-tat-psp-DT | Spe1 | BufferM | MilliQ | total |
---|---|---|---|---|
2 | 0.5 | 1 | 6.5 | 10 |
J2-tat-psp-DT | Pst1 | BufferH | MilliQ | total |
---|---|---|---|---|
2 | 0.5 | 1 | 6.5 | 10 |
at 37℃, 2hours
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-torAGFP-DT 6: 37.4μg/mL, (260/280): 2.24, (280/230): 3.27
Electrophoresis
LaneA: J2-tat (9/6)
LaneB: J2-tat-psp-DT 3C5 (9/13)
LaneC: J2-tat-psp-DT 1C5 (9/15)
LaneD: J2-tat-psp-DT (9/15)
LaneE: J2-tat-psp-DT 1C3 (9/18)
LaneF: J2-tat-psp-DT 3C5 (9/18)
LaneG: J2-tat-psp-DT-LacP-torA-GFP-DT:2 (9/20)
LaneH: J2-tat-psp-DT-LacP-torA-GFP-DT:3 (9/20)
LaneA~H are (E,P)
Restriction enzyme processing
J2-tat-psp-DT-LacPtorA-GFP-DT:2 | BufferH | Pst | MilliQ | total |
---|---|---|---|---|
15 | 3 | 1 | 12 | 30 |
J2-tat-psp-DT-LacP-torAGFP-DT:3 | BufferH | Pst | MilliQ | total |
---|---|---|---|---|
15 | 3 | 1 | 12 | 30 |
Restriction enzyme processing
LacP-torAGFP-DT(37.4μg/mL) | BufferH | EcoR1 | Pst | total |
---|---|---|---|---|
25 | 3 | 1 | 1 | 30 |
Electrophoresis
1: J2-tat-psp-DT-LacP-torA-GFP-DT:2
2: LacP-torA-GFP-DT (E,P) (9/22)
3: J2-tatpsp-DT-LacP-torA-GFP-DT:3
September 23
Electrophoresis
写真お願いします。
- LacP-torA-GFP-DT(EcoR1, Pst1)
lane_1 1kb ladder lane_3 DNA lane_4 DNA lane_5 DNA
Gel extraction
写真お願いします。
- J23107-tatABCD-pspA-DT(pSB1C3)(Spe1, Pst1)
- LacP-torA-GFP-DT(Xba1, Pst1)
lane_1 1kb ladder lane_3 J23107-tatABCD-pspA-DT(pSB1C3)(Spe1, Pst1) lane_5 LacP-torA-GFP-DT(Xba1, Pst1)
Restriction
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
写真お願いします。
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
写真お願いします。
- 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
写真お願いします。
J23107-tatABCD-pspA-DT(pSB1C3) (1-6)
lane_1 1kb ladder
lane_2~7 DNA
lane_8 1kb ladder