Team:HokkaidoU Japan/Notebook/aggregation Week 6
From 2012.igem.org
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#Remove supernatant and air drying in room temperature then added 10 ul of DW. | #Remove supernatant and air drying in room temperature then added 10 ul of DW. | ||
</p> | </p> | ||
- | ==== | + | |
+ | ==digestion== | ||
+ | <p> | ||
+ | digestion of ethanol precipitation product(pBad-RBS). | ||
+ | {|class="hokkaidou-table-digestion" | ||
+ | |- | ||
+ | |DNA solution | ||
+ | |3 ul | ||
+ | |- | ||
+ | |SpeI | ||
+ | |1 ul | ||
+ | |- | ||
+ | |10xM buffer | ||
+ | |1 ul | ||
+ | |- | ||
+ | |DW | ||
+ | |5 ul | ||
+ | |- | ||
+ | |Total | ||
+ | |10 ul | ||
+ | |} | ||
+ | </p> | ||
+ | |||
</div><div> | </div><div> | ||
Revision as of 11:15, 6 August 2012
Contents |
August 6th
Ethanol precipitation
Ethanol precipitation for digestion and gel extraction product.
- Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
- Centrifuged in 15000 rpm, 10 min at 4C.
- Remove supernatant and added 220 ul of 70% ethanol.
- Centrifuged in 15000 rpm, 10 min at 4C.
- Remove supernatant and air drying in room temperature then added 10 ul of DW.
Ligation
We ligated pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.
pT7-RBS | 1 ul |
Ag43-dT | 2 ul |
DW | 2 ul |
Ligation Mighty Mix(TAKARA) | 5 ul |
Total | 10 ul |
Ligation reaction time was written below.
Degree | Minute |
16 | 30 |
65 | 10 |
4 | Hold |
mini-prep
mini-prep of pBad-RBS. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.
transformation
Transformation of plasmid DNA (pT7-RBS-Ag43-dT on pSB1K3)
in E. coli(DH5α).
- Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
- Incubated on ice for 30 min.
- Added 600 ul of LB then incubated the cells for 2 hours at 37C.
- Prepared and Labeled two LBK plates.
- Plated 300 ul of the culture onto first dish and spread.
- Added 900 ul of LB to 100 ul of the culture and plated 300 ul of it onto second dish and spread.
- Incubated them at 37C for hours.
Ethanol precipitation
Ethanol precipitation for mini-prep product (pBad-RBS). Because the refine of mini-prep product is not enough.
We use 15 ul DNA solution.
- Added 1.5 ul of NaoAc(3 M), 1.5 ul of glycogen and 38 ul of 100% ethanol.
- Centrifuged in 15000 rpm, 10 min at 4C.
- Remove supernatant and added 220 ul of 70% ethanol.
- Centrifuged in 15000 rpm, 5 min at 4C.
- Remove supernatant and air drying in room temperature then added 10 ul of DW.
digestion
digestion of ethanol precipitation product(pBad-RBS).
DNA solution | 3 ul |
SpeI | 1 ul |
10xM buffer | 1 ul |
DW | 5 ul |
Total | 10 ul |