Team:HokkaidoU Japan/Notebook/Week 2
From 2012.igem.org
(→July 11th) |
|||
Line 283: | Line 283: | ||
==July 11th== | ==July 11th== | ||
<div class="hokkaidou-notebook-daily"> | <div class="hokkaidou-notebook-daily"> | ||
- | <!-- 峯さんへ | + | |
+ | <!-- 峯さんへ この行を削除して、ここから編集を開始してください。 --> | ||
+ | |||
</div> | </div> |
Revision as of 12:09, 11 July 2012
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.
PCR reaction solution
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.
Electrophoresis resulsts
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