Team:HokkaidoU Japan/Notebook/aggregation Week 3

Ag43, dT

Electrophoresis

Results of digestion at 15th.

Product:Ag43(K346007)=3120bp and 2070bp, pT7-RBS on pSB1K3=2247bp
We confirmed there are some kinds of restriction enzyme site in K346007 (digested with SpeI, PstI), and pT7-RBS on pSB1K3 was successfully digested with EcoRI and PstI.　Balance between d+(EcoRI) and d+(E&P:cut with EcoRI and PstI) is about 80bp.

Gel extraction

Gel extraction for digestion products. Used FastGene Gel&PCR extraction kit(NipponGenetics) and　got 50 ul of DNA solution.

Ethanol precipitation

Ethanol precipitation for digested and extracted products.

1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
2. Centrifuged at 15000 rpm, 10 min at 4C.
3. Remove supernatant and added 220 ul of 70% ethanol.
4. Centrifuged at 15000 rpm, 10 min at 4C.
5. Remove supernatant and air drying at room temperature then added 5 ul of DW.

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dT(B0015) would be amplified　incorrectly and we couldn't get enough digestion product. So we tried another DNA amplification method: PCR then digested.

PCR

PCR for dT(B0015)

DNA solution	1 ul
KOD-Plus-NEO(Taq polymerase)	1 ul
dNTP	5 ul
MgSO4	3 ul
KOD-Plus-NEO Buffer	5 ul
Forward Primer(100bp_up forward primer)	1 ul
Reverse Primer(200bp_down Reverse primer)	1 ul
DW	33 ul
Total	50 ul

Number	Degree	Second
1	94	120
2	98	10
3	58	30
4	68	30
5	4	HOLD

Cycle:2~4 x 45

We migrated B0015 of mini-prep product, digestion product, and PCR product. dT done PCR would have 429bp(100bp(added by forward primer) + 129bp(dT) + 200bp(added by reverse primer)). Most bright and thick band in this image has about 300~400 bp. We thought dT was amplified successfully.

Gel extraction

Gel extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics). Got 50 ul of DNA solution.

Digestion

Digestion for dT which amplified with PCR. Digested with XbaI and PstI. dT

DNA solution	5 ul
XbaI	1 ul
PstI	1 ul
10xM buffer	2 ul
DW	11 ul
Total	20 ul

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Ag43

Digestion result of Ag43 was incorrect. We digested Ag43 once more time.

Digestion

Digestion for Ag43 with SpeI and PstI.

Ag43 DNA solution	9 ul
SpeI	1 ul
PstI	1 ul
10xH buffer	2 ul
DW	7 ul
Total	20 ul

There are same results with digestion result of recent. We thought PstI would cut different site. What is this 500bp fragment????

Gel extraction

Gel extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics). Got 50ul of DNA solution.

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Liquid Culture

Liquid culture for single colony isolated pT7-RBS on pSB1C3 and Ag43-dT on pSB1T3. Colonies which have Ag43-dT on pSB1T3 were closely existed so we would picked up two or more colonies.

1. Picked up one (or tow?) colony from single colony isolated plates　by platinum loop.
2. Dipped into 2 ml of LBC and LBT.
3. Incuvated.

Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3

Plasmid extraction

Plasmid extraction for Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 liquid culture products incuvated from yesterday(16th). We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

Electrophoresis

Electrophoresis for digestion product of K346007(Ag43) with EcoRI and SpeI as a reference to confirm SpeI cuts correct site or not and PstI didn't work correctly(in past experiments). And mini-prep products of Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 also migrated. If digestion were succeeded, there are two bands would be seen: about 3120bp and 2070bp. And if ligation-transformation-mini-prep were succeeded, there would be existed two bands in each lanes: about 5000bp and 2000bp.

From this result we confirmed Ag43 was successfully digested with EcoRI and SpeI, and there were no other extra bands which had been existed double digested with EcoRI & PstI and SpeI & PstI. would PstI not work correctly?

We planed to use dT as vector and Ag43 as Insert. A problem is if dT on pSB1AK3 is used as vector and Ag43 used as insert, we can't select correct Ag43 band in gel extraction phase because DNA bp of Ag43 is nearly same as pSB1AK3. Thus we tried to use dT on pSB1T3 as vector and Ag43 on pSB1C3 as vector and ligate with standard assembly.

About mini-prep products, in Ag43-dT on pSB1T3, there were two plasmid DNA bands about 8000bp and 3000bp in one lane. We thought which means we failed single colony isolation then resuspended two another E.coli colonies another ligated DNA were transformed. In pT7-RBS on pSB1C3, we needed about 2000bp plasmid DNA and there were about 1500bp of DNA. Plasmid DNA migrates more far than linear DNA so we thought we got correct DNA.

To confirm mini-prep products were really ligated correct DNA fragments, first we extracted Ag43-dT on pSB1T3(low bp band) from TBE gel and digested with EcoRI and PstI.

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dT Vector plasmid change To use dT as a vector, we needed to change plasmid backbone pAB1AK3 to pSB1T3.

Digestion

Digestion to change the plasmid backbone. Used DNA solution as PCR product(did at 16th) and digestioned pSB1T3 were already exist.

Digestion mix

double digestion(EcoRI and PstI)

dT PCR product	1 ul
EcoRI	1 ul
PstI	1 ul
10xH buffer	2 ul
DW	15 ul
Total	20 ul

Control 1(EcoRI only)

dT PCR product	1 ul
EcoRI	1 ul
10xH buffer	2 ul
DW	16 ul
Total	20 ul

Control 2(PstI only)

dT PCR product	1 ul
PstI	1 ul
10xH buffer	2 ul
DW	16 ul
Total	20 ul

We couldn't confirm digestion results.

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pT7-RBS on pSB1C3 and Ag43-dT on pSB1T3

Electrophoresis and gel extraction

Gel extraction for Ag43-dT on pSB1T3.
We cut low bp band (see image below). Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics). Got 50 ul of DNA solution.

Digestion

Digestion Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 to confirm ligation was succeeded or not.

Ag43-dT on pSB1T3(30 ng/ul)

Control 1(EcoRI only)

Ag43-dT DNA solution	3.3 ul
EcoRI	1 ul
10xH buffer	2 ul
DW	14.7 ul
Total	21 ul

Control 2(PstI only)

Ag43-dT DNA solution	3.3 ul
PstI	1 ul
10xH buffer	2 ul
DW	14.7 ul
Total	21 ul

double digestion(EcoRI & PstI)

Ag43-dT DNA solution	3.3 ul
EcoRI	1 ul
PstI	1 ul
10xH buffer	2 ul
DW	13.7 ul
Total	21 ul

pT7-RBS on pSB1C3(40 ng/ul)

Control 1(EcoRI only)

pT7-RBS DNA solution	2.5 ul
EcoRI	1 ul
10xH buffer	2 ul
DW	15.5 ul
Total	21 ul

Control 2(PstI only)

pT7-RBS DNA solution	2.5 ul
PstI	1 ul
10xH buffer	2 ul
DW	15.5 ul
Total	21 ul

double digestion(EcoRI & PstI)

pT7-RBS DNA solution	2.5 ul
EcoRI	1 ul
PstI	1 ul
10xH buffer	2 ul
DW	14.5 ul
Total	21 ul

From this digestion results,

• Cut with EcoRI,PstI and E&P(EcoRI and PstI) showed different base pair bands compared with d-(digestion minus).
• Cut with E&P showed two DNA bands: about 2000bp and 500~1000 bp.
• All of digestion products existed lower place than correct band.Correct DNA would show about 5000bp(EcoRI and PstI), 3000bp and 2000bp (E&P).

We decided change plasmid backbone of dT pSB1AK3 to pSB1T3.
If we cut Ag43-dT on pSB1AK3 with EcoRI and PstI to confirm insert DNA, DNA fragment base pair resemble each other(about 3000bp), so then we can't identify them.

digestion

Digestion to confirm what kind of restriction enzyme sites Ag43(K346007) has.

EcoRI

DNA solution	1 ul
EcoRI	1 ul
10xH buffer	1 ul
DW	7 ul
Total	10 ul

XbaI

DNA solution	1 ul
XbaI	1 ul
10xM buffer	1 ul
BSA	1 ul
DW	6 ul
Total	10 ul

SpeI

DNA solution	1 ul
SpeI	1 ul
10xH buffer	1 ul
DW	7 ul
Total	10 ul

PstI

DNA solution	1 ul
PstI	1 ul
10xH buffer	1 ul
DW	7 ul
Total	10 ul

From this result, we doubted are there one or more another PstI resutriction enzyme site except BioBrick suffix?

PCR

PCR to confirm what DNA fragment is ligated to vector as insert. We used PCR for pT7-RBS on pSB1C3. So the result would be 80~90bp band.

DNA solution	1 ul
KOD-Plus-NEO(Taq polymerase)	1 ul
dNTP	5 ul
MgSO4	3 ul
KOD-Plus-NEO Buffer	5 ul
Forward Primer(Biobrick prefix forward primer)	1 ul
Reverse Primer(Biobrick suffix Reverse primer)	1 ul
DW	33 ul
Total	50 ul

Number	Degree	Second
1	94	120
2	98	10
3	68	60
4	4	HOLD

Cycle:2~4 x 45

Liquid Culture

Liquid culture for single colony isolated(19hrs incubated) Ag43-dT on pSB1T3.

1. Prepared 2 ml LB.
2. Added Tet.
3. Resuspended one colony.
4. Incuvated 16 hrs.

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We decided to start another ligation plan. First we digestion Ag43(K346007) and dT(B0015) with EcoRI&SpeI and EcoRI&XbaI then ligate. Ligation product cut with XbaI&SpeI and HindiIII. HindiIII can cut the site which is exist in pSB1AK3 then we can confirm Ag43-dT(about 3000bp) and pSB1K3(3000bp: cutting fragment will be about 1000bp). Next we digest pT7-RBS on pSB1C3 with SpeI only and ligate X-Ag43-dT-S and S-pSB1C3-pT7-RBS-S. If that go well, we can get pT7-RBS-Ag43-dT on pSB1C3 plasmid DNA which has never been digested with PstI.

Liquid culture

Liquid culture for Ag43(K346007)

Plasmid extraction

Plasmid extraction for Ag43-dT on pSB1T3 and Ag43(K346007) which is inserted in pSB1C3. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

Electrophoresis

Electrophoresis for Ag43-dT on pSB1T3[about 5400bp] and Ag43(K346007)[about 5200bp] mini-prep products.

From this mini-prep result, Ag43-dT on pSB1T3 showed obviously higher DNA band than we estimated. It means Ag43-dT and pSB1T3 were misligated. And Ag43(K346007), we thought Ag43(K346007) DNA band existed in correct area because this DNA has about 5200bp and plasmid DNA migrates more far than linear DNA. Additionally, one weak 10kbp band existed in Ag43(K346007) lane.

Digestion

We conducted two digestion.

• Digestion for Ag43(K346007) cut with EcoRI and SpeI. This DNA is for confirmation of PstI restriction enzyme cutting site.
• Digestion for Ag43(K346007) and dT(B0015) cut with EcoRI & SpeI and EcoRI & XbaI.

But we cut dT PCR product which didn't have plasmid vector so we retry digestion of dT after.

Insert Ag43(K346007)(E&S)

DNA solution	16 ul
EcoRI	1 ul
SpeI	1 ul
10xH buffer	2 ul
Total	20 ul

Vector dT(E&X)

DNA solution	4 ul
EcoRI	1 ul
XbaI	1 ul
10xM buffer	2 ul
DW	12 ul
Total	20 ul

dT(EcoRI)

DNA solution	4 ul
EcoRI	1 ul
10xH buffer	2 ul
DW	13 ul
Total	20 ul

dT(XbaI)

DNA solution	4 ul
XbaI	1 ul
10xM buffer	2 ul
DW	13 ul
Total	20 ul

Ag43(K346007)(E&S)

DNA solution	10 ul
EcoRI	1 ul
SpeI	1 ul
10xH buffer	2 ul
DW	7.8 ul
Total	20 ul

From this result, we confirmed Ag43(K346007) was successfully digested into two fragments and low concentration of restriction enzyme cause unperfectly cut of mini-prep product. But there are correct two bands in Ag43 cutting result so restriction enzyme worked. And about dT digestion, XbaI worked in halfway. We thought this is because we didn't added BSA buffer into dT digestion mix.

And we retried digestion of dT which was mini-prep and gel extracted products. Recipe was same as above(E&X).

Gel extraction

Gel extraction for Ag43(K346007) digestion products.We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

Electrophoresis

Electrophoresis for digestion result done yesterday(dT cut with EcoRI and XbaI). Added 5 ul of EtBr and Pre-migrated for 30 min. Additionaly, to confirm the concentration of Ag43(K346007) gel extract result.

From this result, we confirmed dT was successfully cut with EcoRI & SpeI.

Gel extraction

Gel extraction for Ag43(K346007) digestion products.We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

Ethanol precipitation

Ethanol precipitation for gel extraction products above.

1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
2. Centrifuged at 15000 rpm, 10 min at 4C.
3. Removed supernatant and added 220 ul of 70% ethanol.
4. Centrifuged at 15000 rpm, 5 min at 4C.
5. Remove supernatant and dried out at room temperature after that added 5 ul of DW.

Ligation

We ligated Ag43(purified at 7/17 and 7/20) as an insert and dT as vector.

Ag43(7/20) + dT

Ag43	2 ul
dT	2 ul
DW	1 ul
Ligation Mighty Mix(TAKARA)	5 ul
Total	10 ul

Ag43(7/17) + dT

Ag43	4 ul
dT	2 ul
Ligation Mighty Mix(TAKARA)	6 ul
Total	12 ul

Ligation reaction time was written below.

Degree	Minute
16	30
65	10
4	Hold

Transformation

Electrophoresis

Electrophoresis for digestion and ligation products yesterday.

There are three bands in ligation products(Ag43(7/20) + dT(on pSB1AK3))). Lower band would be digestion result which couldn't be ligated with dT. Middle band would be successfully ligaed DNA which have about 6k bp(Ag43 has 3.1k bp and dT on pSB1AK3 has 3.2k bp). And higher band would make something dimer, we thought.

Single colony isolation

Single colony isolation for incubated colonies spread yesterday.

Ethanol precipitation

Ethanol precipitation for gel extracted Ag43(E&S) gel extraction product for digestion of PstI Star activity confirmation.

1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
2. Centrifuged at 15000 rpm, 10 min at 4C.
3. Removed supernatant and added 220ul of 70% ethanol.
4. Centrifuged at 15000 rpm, 10 min at 4C.
5. Removed supernatant and dried out at room temperature after that added 10 ul of DW.

Electrophoresis

Electrophoresis to confirm the concentration of DNA solution.

Digestion

Digestion to confirm there are some PstI cutting site in Ag43(K346007)(cut with EcoRI and SpeI to remove PstI this parts potentially has).

Used DNA	K346007 cut with EcoRI and SpeI
Concentration(ng/ul)	25
Used DNA volume(ul)	2
theoretical ez value(ul)	0.02

PstI = 0.1ul

DNA solution	2 ul
PstI	0.1 ul
10xH buffer	1 ul
DW	6.9 ul
Total	10 ul

PstI = 0.2 ul

DNA solution	2 ul
PstI	0.2 ul
10xH buffer	1 ul
DW	6.8 ul
Total	10 ul

PstI = 0.5 ul

DNA solution	2 ul
PstI	0.5 ul
10xH buffer	1 ul
DW	6.5 ul
Total	10 ul

PstI = 1.0 ul

DNA solution	2 ul
PstI	1 ul
10xH buffer	1 ul
DW	6 ul
Total	10 ul

From this digestion, we confirmed there is at least one PstI cutting site in Ag43 fragment because there digestion results showed same bp and same number of cutting band.

PCR

PCR to confirm how ligate Ag43 fragment(s) in dT on pSB1AK3. We used Ag43 last 700bp area as primer which had designed as sequencing primer. If three (two forward and one reverse )Ag43 were inserted, this primer can anneal to forward Ag43 as forward primer and reverse Ag43 as reverse primer then amplify about 766bp. If there are no reverse Ag43, DNA can't be amplified. PCR recipe

DNA solution	1 ul
KOD-Plus-NEO(Taq polymerase)	1 ul
dNTP	5 ul
MgSO4	3 ul
KOD-Plus-NEO Buffer	5 ul
Ag43-forward primer	2 ul
DW	33 ul
Total	50 ul

Number	Degree	Second
1	94	120
2	98	10
3	58	60
4	4	HOLD

Cycle:2~4 x 45

We failed amplification. Retry!

PCR

We did PCR written above once again. Change some point of reaction. We amplified Ag43(K346007) original DNA also as a control. PCR recipe

DNA solution	2 ul
KOD-Plus-NEO(Taq polymerase)	1 ul
dNTP	5 ul
MgSO4	3 ul
KOD-Plus-NEO Buffer	5 ul
Ag43-forward primer	2 ul
DW	33 ul
Total	50 ul

Number	Degree	Second
1	94	120
2	98	10
3	58	30
4	68	30
5	4	HOLD

Cycle:2~4 x 45

Electrophoresis

Results of digestion at 21th.
We expected appear the band of about 700bp. So, we use 1% and 2% agarose gel.

We can't find some band in 700~800bp...

Liquid Culture

Liquid culture for single colony isolated Ag43-dT on pSB1AT3.

1. Picked up one colony from single colony isolated plates　by platinum loop.
2. Dipped into 2 ml of LBA.
3. incubated for 16 hrs.

Digestion

Digested Ag43(K346oo7) mini-prep product with EcoRI and SpeI.

DNA solution	5 ul
EcoRI	1 ul
SpeI	1 ul
10xH buffer	2 ul
DW	11 ul
Total	20 ul