Team:HokkaidoU Japan/Notebook/aggregation Week 2

pT7 + RBS (3A Assembly) and Ag43 + dT (standard assembly) of ligation products were transformed and incubated. We confirmed ideal insert DNA (pT7 and RBS or Ag43 and dT) were inserted by colony PCR.

Colony PCR

Colony PCR for assembly products.

1. Picked up colony from LB plates by Autoclaved toothpicks.
2. Re-suspension into 10 ul DW in 1.5 ml eppendorf tubes.
3. 4 ul was added in PCR reaction solution, and 6 ul was mixed with 200 ul LB.
4. Ran PCR machine in recipe below.

PCR reaction solution

PCR recipe (pT7 + RBS)

Cycle:2~3 x 40

(Ag43 + dT) Ag43 + dT (+ Biobrick prefis & suffix) is about 3290bp.

Cycle:2~3 x 35

Electrophoresis results

PCR result

PCR result

Electrophoresis for (pT7 + RBS) by 2% agarose gel and (Ag43 + dT) by 1% agarose gel.
pT7 + RBS on pSB1K3 bbp-Insert-bbs:86bp

Ag43 + dT on pSB1AK3 bbp-Insert-bbs:3290bp

We couldn't confirm insert DNA were really ligated with Vector or not. Next step, we tried confirmation of insert DNA by electrophoresis of extracted plasmids. For plasmid extraction, we needed to incubate in liquid culture.

Incubate for plasmid extraction

1. Prepared 1800 ul LB solutions.
2. Mixed 200 ul of cultures (suspention were incubated for 2 hrs) and antibiotics(Km (pT7-rbs on pSB1K3), Amp(Ag43-dT on pSB1AK3)).
3. Incubated for 15 hrs and 30 min.

Plasmid extraction

Extracted plasmids from some colonies (we selected colonies which showed more correct bands than other colonies, pT7+RBS were No. 4, 5, 9, 10 colonies and Ag43 + dT were No. 1, 2, 3, 4 colonies)incubated in LBA(Ag43 + dT) and LBK(pT7 + RBS) for 15 hrs and 30 min.

1. Extracted from LB culturing products by using FastGene Plasmid Mini kit(Nippon Genetics).
2. Electrophoresis (Used pre-migrated 1% agarose gels with 5 ul EtBr)in 30 min.

pT7 + RBS on pSB1K3(Total 2247bp)

Electrophoresis resulsts

Ag43 + dT on pSB1AK3(Total 6444bp)

Electrophoresis resulsts

To confirm more correctly about insert DNA, we tried to digest these DNA with EcoRI and PstI.

Digestion

Digestion for confirmation of insert DNA. Each exracted DNA were digested with E & P.
Digestion recipe
pT7-RBS

Digestion recipe
Ag43-dT

Digestioned at 37c for 2 hrs.

pT7-RBS digestion results

Ag43-dT digestion results

Insert DNA were correct we thought. We tried digestion for 3A Assembly[pT7-RBS+Ag43-dT+pSB1C3]

Digestion

Digestion for 3A Assembly.
pT7-RBS

Ag43-dT

pSB1C3

Reacted for 2 hrs at 37c.

Ligation

Ligation of pT7-RBS + Ag43-dT + pSB1C3(3A Assembly)
Ligation recipe

Ligation reaction time was written below.

ligation result

Transformation

Transformation for pT7-RBS + Ag43-dT + pSB1C3 into BL21(DE3)(E.coli which have T7 polymerase coading site in their genome).

1. Added DNA solutions (Ligation products) 1 ul to compitent cell of BL21(DE3).
2. Incubated on ice for 30 min.
3. Heatshock for 1 min at 42c.
4. Added 200 ul of LB to transformed BL21(DE3) solution.
5. Pre-incubate for 2 hrs
6. Spread 200 ul of supernant of LB&BL21(DE3) solution to LBC plate.
7. 50ul of LB&BL21(DE3) solution was added to 200ul LB solution then spread 200 ul to LBC plate.
8. Incubated for 23 hrs and 30 minutes.

Colony PCR

Colony PCR for ligation product.Each products were reacted in recipes written below.

1. Picked up each 16 colonies from LBC plates by Autoclaved toothpicks.
2. Dipped into 10 ul DW in 1.5 ml eppendorf tubes.
3. From 10 ul DW, 4 ul was added in PCR reaction solution (below), 6 ul was mixed with 200 ul LB.
4. Ran PCR machine in recipe below.
5. Electrophoresis for confirmation of PCR results.

PCR reaction solution

PCR recipe

(pT7 + RBS)

Cycle:2~3 x 40

Liquid culture

Liquid culture for some colonies used in colony PCR.

1. Prepared 200 ul LB solutions.
2. To these LB solutions, added 6 ul of LB solutions (colony PCR solutions were pre-incubated for 3 hrs) and added 2 ml LB and antibiotic(Cp).
3. Incubated for 18 hrs and 30 min.

Plasmid extraction

Plasmid extraction from colony No. 1~8.
We used mini-prep kit FastGene Plasmid Mini Kit (Nippon Genetics), and got 50 ul of DNA solution.

Plasmid extraction results

Digestion

Digestion to confirm how DNA fragments ligated.
Digestion Recipe

Digestion results

Ligation

Ligation for digestion fragments written above.
Ligation recipe
Each DNA fragments (pT7-RBS + pSB1C3 and Ag43-dT + pSB1T3) were reacted in recipe written below.

We tried electrophoresis of colony no. 1 (see colony pcr result at 9th) from ligation product.

ligation product

Transformation

Transformation for ligation products written above.

1. Added DNA solutions (Ligation products) 1 ul to DH5α compitent cell.
2. Incubated on ice for 30 min.
3. Added 600 ul of LB to transformed DH5α solution.
4. Pre-incubate for 2 hrs
5. Spread 300 ul of LB&DH5α solution to LBC and LBT plate, and 100 ul was added into 900 ul of LB.
6. Spread 300 ul of LB&DH5α solution from 1000 ul LB (made at 5) to LBC and LBT plate.
7. Incubated for 21 hrs.

And to confirm success of ligation about pT7-RBS-Ag43-dT on pSB1C3, we tried to digest this DNA with EcoRI and PstI once more time. Showed the result.

digestion results

Liquid culture

Liquid culture for Ag43(BBa_K346007)

1. Picked up one colony from plate done single colony isolation.
2. Dipped into LBC solution.
3. Incubated for 16 hrs.

Gel extraction

Used Gel Extraction Kit (FastGene Gel/PCR extraction kit:NipponGenetics) to extract digestion products (see July 14th).

Digestion

Digestion to confirm that what kind of restriction enzyme cutting sites are there in DNA (colony 1 and 6).
EcoRI

XbaI

PstI

SpeI

EcoRI + PstI

XbaI + SpeI

Ethanol precipitation

Ethanol precipitation for digestion products.

1. Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 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. Removed supernatant and dried out at room temperature, after that added 5 ul of DW.

Electrophoresis

Electrophoresis for digest-ethanol precipitation products.

1. Added 5 ul of EtBr.
2. Migrated for 30 min.

digestion results

Single colony isolation

Single colony isolation for transformation products of yesterday.

1. Picked up one colony from incubated LBC and LBT plate.
2. Spread it on LBC and LBT plate.
3. Incubated for 16 hrs.

Digestion

Digestion of Ag43(with S,P), dT(With X,P) and pT7-RBS(With E).

Ag43

dT

pT7-RBS