Team:HokkaidoU Japan/Notebook/aggregation Week 2
From 2012.igem.org
Contents |
July 9th
pT7 + RBS (3A Assembly) and Ag43 + dT (standard assembly) ligation products were transformed and cultivated then some colonies were formed (12~16 colonies) so we confirmed really insert DNA (3A:pT7 and RBS, standard:Ag43 and dT) were inserted to vector or not by colony PCR.
Colony PCR
Colony PCR for assembly products.Each product reacted recipes written below.
- picked up each 12(16 is best but there were only 12 colonies) colonies from LB plates by Autoclaved toothpicks.
- Dipped into 10ul DW in 1.5ml eppendorf tubes.
- from 10ul DW, 4ul was added in PCR reaction solution (below), 6ul was mixed with 200ul LB.
- Ran PCR machine in recipe below.
- Electrophoresis for confirmation of PCR results.
DNA solution | 4ul |
KapaTaq ready mix | 5ul |
BioBrick prefix forward primer | 0.5ul |
BioBrick suffix reverse primer | 0.5ul |
Total | 10ul |
PCR recipe
(pT7 + RBS)
Number | Degree | Second |
1 | 94 | 120 |
2 | 94 | 30 |
3 | 68 | 60 |
4 | 4 | HOLD |
Cycle:2~3 x 40 (Ag43 + dT) Ag43 + dT (+ Biobrick prefis & suffix) is about 3290bp. Extension step needed >180seconds.
Number | Degree | Second |
1 | 94 | 120 |
2 | 94 | 30 |
3 | 68 | 180 |
4 | 4 | HOLD |
Cycle:2~3 x 35
Electrophoresis results
Electrophoresis for (pT7 + RBS) in 2% agarose gel and (Ag43 + dT) in 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 we tried confirmation of insert DNA by Electrophoresis of mini-prep products.
For mini-prep, we needed do liquid culture.
Liquid culturing
Liquid culture for mini-prep((pT7 + RBS) on pSB1K3 and (Ag43 + dT) on pSB1AK3).
- Prepared 1800ul LB solutions.
- To these LB solutions, added 200ul of LB solutions (colony PCR solutions were pre-cultivated in about 2hrs) and each antibiotics(Km (for (pT7 + RBS) on pSB1K3) and Amp(for (Ag43 + dT) on pSB1AK3)).
- Cultivated 15hrs30min.
July 10th
Mini-prep
Mini-prep for 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)cultivated in LBA(Ag43 + dT) and LBK(pT7 + RBS) in 15hrs 30min.
- Mini-prep for LB culturing products along the protocol of FastGene Plasmid Mini kit(Nippon Genetics).
- Electrophoresis (Used pre-migrated 1% agarose gels with 5ul EtBr)in 30min.
pT7 + RBS on pSB1K3(Total 2247bp)
Ag43 + dT on pSB1AK3(Total 6444bp)
To confirm more about insert DNA, we tried to digest these DNA with EcoRI and PstI.
Digestion
Digestion for confirmation of insert DNA. Each DNA mini-prep products were digested with E & P. Digestion recipe pT7-RBS
pT7-RBS | 1,5ul |
EcoRI | 1ul |
PstI | 1ul |
10xH buffer | 2ul |
DW | 14.5ul |
Total | 20ul |
Digestion recipe
Ag43-dT
Ag43-dT | 4ul |
EcoRI | 1ul |
PstI | 1ul |
10xH buffer | 2ul |
DW | 12ul |
Total | 20ul |
Digestioned at 37c in 2hrs.
Digestion results
pT7+RBS
Ag43+dT
Insert DNA were correct we thought. We tried digestion for 3A Assembly[pT7-RBS + Ag43-dT + pSB1C3]
Digestion
Digestion for 3A Assembly. pT7-RBS
DNA | 17ul |
EcoRI | 1ul |
SpeI | 1ul |
10xH buffer | 3ul |
DW | 8ul |
Total | 30ul |
Ag43-dT
DNA | 12.5ul |
XbaI | 1ul |
PstI | 1ul |
10xH buffer | 2ul |
DW | 3.5ul |
Total | 20ul |
pSB1C3
DNA | 20ul |
EcoRI | 1ul |
PstI | 1ul |
10xH buffer | 3ul |
DW | 5ul |
Total | 30ul |
Reacted in 2hrs at 37c.
July 11th
Ligation
Ligation of pT7-RBS + Ag43-dT + pSB1C3(3A Assembly) Ligation recipe
pT7-RBS | 2ul |
Ag43-dT | 2ul |
pSB1C3 | 3ul |
Ligation Mighty Mix(TAKARA) | 8ul |
Total | 16ul |
Ligation reaction time was written below.
Degree | Minute |
16 | 30 |
65 | 10 |
4 | Hold |
Electrophoresis result
Transformation
Transformation for pT7-RBS + Ag43-dT + pSB1C3 into BL21(DE3)(E.coli which have T7 polymerase coadin site in their genomic DNA).
- Added DNA soltions (Ligation products) 1ul to BL21(DE3) compitent cell.
- Stood on ice in 30min.
- Heatshock for 1min at 42c.
- Added 200ul of LB to transformed BL21(DE3) solution.
- Pre-cultivate in 2hrs
- Splead 200ul of LB&BL21(DE3) solution supernant to LBC.
- 50ul of LB&BL21(DE3) solution were added to 200ul LB solution then spread 200ul to LBC plate.
- Cultivated.
July 12th
Colony PCR
Colony PCR for ligation product.Each products were reacted in recipes written below.
- picked up each 16 colonies from LB plates by Autoclaved toothpicks.
- Dipped into 10ul DW in 1.5ml eppendorf tubes.
- from 10ul DW, 4ul was added in PCR reaction solution (below), 6ul was mixed with 200ul LB.
- Ran PCR machine in recipe below.
- Electrophoresis for confirmation of PCR results.
DNA solution | 4ul |
KapaTaq ready mix | 5ul |
BioBrick prefix forward primer | 0.5ul |
BioBrick suffix reverse primer | 0.5ul |
Total | 10ul |
PCR recipe
(pT7 + RBS)
Number | Degree | Second |
1 | 94 | 120 |
2 | 94 | 30 |
3 | 68 | 90 |
4 | 4 | HOLD |
Cycle:2~3 x 40
Liquid culturing
Liquid culture for some colonies used in colony PCR.
- Prepared 200ul LB solutions.
- To these LB solutions, added 6ul of LB solutions (colony PCR solutions were pre-cultivated in about 3hrs) and added 2ml LB and antibiotic(Cp).
- Cultivated 18hrs30min.
July 13th
July 14th
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.
Insert | 5ul |
Vector | 1ul |
Ligation Mighty Mix(TAKARA) | 6ul |
Total | 12ul |
Degree | Minute |
16 | 30 |
65 | 10 |
4 | Hold |
Ligation result with colony no.1 (see colony pcr result in 9th)
Transformation
Transformation for ligation products written above.
- Added DNA soltions (Ligation products) 1ul to DH5α compitent cell.
- Stood on ice in 30min.
- Added 600ul of LB to transformed DH5α solution.
- Pre-cultivate in 2hrs
- Splead 300ul of LB&DH5α solution to LBC and LBT , 100ul added into 900ul of LB.
- Splead 300ul of LB&DH5α solution from 1000ul LB (100ul added into 900ul) to LBC and LBT.
- Cultivated in 21hrs.
Liquid culture
Liquid culture for Ag43(BBa_K346007)
- Picked up one colony from single colony isolated plate.
- Dipped into LBC.
- cultivated.
July 15th
Gel extraction
We used Gel Extraction Kit (FastGene Gel/PCR extraction kit:NipponGenetics) to purify digestion products (see 14th).
Digestion
Digestion to confirm what kind of restriction enzyme cutting sites these DNA (colony 1 and 6) have. EcoRI
DNA solution | 6ul |
EcoRI | 1ul |
10xH buffer | 1ul |
DW | 2ul |
Total | 10ul |
XbaI
DNA solution | 6ul |
XbaI | 1ul |
10xM buffer | 1ul |
BSA | 1ul |
DW | 1ul |
Total | 10ul |
PstI
DNA solution | 6ul |
PstI | 1ul |
10xH buffer | 1ul |
DW | 2ul |
Total | 10ul |
SpeI
DNA solution | 6ul |
SpeI | 1ul |
10xM buffer | 1ul |
DW | 2ul |
Total | 10ul |
EcoRI + PstI
DNA solution | 6ul |
EcoRI | 1ul |
PstI | 1ul |
10xH buffer | 1ul |
DW | 11ul |
Total | 20ul |
XbaI + SpeI
DNA solution | 6ul |
XbaI | 1ul |
SpeI | 1ul |
10xM buffer | 1ul |
DW | 11ul |
Total | 20ul |
Ethanol precipitation
Ethanol precipitation for digestion products.
- Added 2ul of NaoAc, 1.5ul of glycogen and 50ul of 100% ethanol.
- Centrifuged in 15000rpm, 10min at 4C.
- Remove supernatant and added 220ul of 70% ethanol.
- Centrifuged in 15000rpm, 5min at 4C.
- Remove supernatant and air drying in room temperature then added 5ul of DW.
Electrophoresis
Electrophoresis for digest-ethanol precipitation products.
- added 5ul of EtBr.
- Migrated in 30min.
Single colony isolation
Single colony isolation for Transformation products synthesized yesterday.
- one colony picked up from cultivated LBC and LBT plate.
- Spread on LBC and LBT.
- Cultivated.
Digestion
Digestion of Ag43(with S,P), dT(With X,P) and pT7-RBS(With E). Ag43
DNA solution | 9ul |
SpeI | 1ul |
PstI | 1ul |
10xH buffer | 2ul |
DW | 7ul |
Total | 20ul |
dT
DNA solution | 1.1ul |
XbaI | 1ul |
PstI | 1ul |
10xM buffer | 2ul |
DW | 14.9ul |
Total | 20ul |
pT7-RBS
DNA solution | 6ul |
EcoRI | 1ul |
10xH buffer | 2ul |
DW | 11ul |
Total | 20ul |