Team:HokkaidoU Japan/Notebook/aggregation Week 7
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+ | <div class="hokkaidou-notebook-daily"> | ||
+ | ==August 16th== | ||
+ | <div> | ||
+ | ==ligation== | ||
+ | <p> | ||
+ | We ligated pBad-RBS on pSB1A3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert. | ||
+ | |||
+ | |||
+ | {|class="hokkaidou-table-ligation" | ||
+ | |- | ||
+ | |pT7-RBS (5 ng/ul) | ||
+ | |1 ul | ||
+ | |- | ||
+ | |Ag43-dT (25 ng/ul) | ||
+ | |2 ul | ||
+ | |- | ||
+ | |Ligation Mighty Mix(TAKARA) | ||
+ | |5 ul | ||
+ | |- | ||
+ | |DW | ||
+ | |2 ul | ||
+ | |- | ||
+ | |Total | ||
+ | |10 ul | ||
+ | |} | ||
+ | |||
+ | |||
+ | Ligation reaction time was written below. | ||
+ | |||
+ | {|class="hokkaidou-table-ligation" | ||
+ | |- | ||
+ | |Degree | ||
+ | |Minute | ||
+ | |- | ||
+ | |16 | ||
+ | |30 | ||
+ | |- | ||
+ | |65 | ||
+ | |10 | ||
+ | |- | ||
+ | |4 | ||
+ | |Hold | ||
+ | |} | ||
+ | </p> | ||
+ | |||
+ | ==Transformation== | ||
+ | <p> | ||
+ | Transformation of plasmid DNA ligation product (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 30min. | ||
+ | #Added 350 ul of LB. | ||
+ | #Prepared and Labeled two petri dishes with LBA. | ||
+ | #Plate 300 ul of the transformation onto first dish and spread. | ||
+ | #Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. | ||
+ | #Incubated the plates at 37C for hours. | ||
+ | </p> | ||
+ | |||
+ | ==Digestion== | ||
+ | <p> | ||
+ | Ag43-dT on pSB1AK3 digestion with SpeI and XbaI. | ||
+ | |||
+ | Ag43-dT | ||
+ | SpeI and XbaI | ||
+ | {|class="hokkaidou-table-digestion" | ||
+ | |- | ||
+ | |DNA solution (100 ng/ul) | ||
+ | |12 ul | ||
+ | |- | ||
+ | |SpeI | ||
+ | |1 ul | ||
+ | |- | ||
+ | |XbaI | ||
+ | |1 ul | ||
+ | |- | ||
+ | |10xH buffer | ||
+ | |2 ul | ||
+ | |- | ||
+ | |DW | ||
+ | |4 ul | ||
+ | |- | ||
+ | |Total | ||
+ | |20 ul | ||
+ | |} | ||
+ | |||
+ | </p> | ||
+ | [[image:|thumb|pBad-RBS on pSB1A3 electrophoresis result]] | ||
+ | [[image:|thumb|digestion result]] | ||
+ | |||
+ | </div><div> | ||
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{{Team:HokkaidoU_Japan/footer}} | {{Team:HokkaidoU_Japan/footer}} |
Revision as of 06:00, 16 August 2012
Contents |
August 16th
ligation
We ligated pBad-RBS on pSB1A3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.
pT7-RBS (5 ng/ul) | 1 ul |
Ag43-dT (25 ng/ul) | 2 ul |
Ligation Mighty Mix(TAKARA) | 5 ul |
DW | 2 ul |
Total | 10 ul |
Ligation reaction time was written below.
Degree | Minute |
16 | 30 |
65 | 10 |
4 | Hold |
Transformation
Transformation of plasmid DNA ligation product (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 30min.
- Added 350 ul of LB.
- Prepared and Labeled two petri dishes with LBA.
- Plate 300 ul of the transformation onto first dish and spread.
- Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread.
- Incubated the plates at 37C for hours.
Digestion
Ag43-dT on pSB1AK3 digestion with SpeI and XbaI. Ag43-dT SpeI and XbaI
DNA solution (100 ng/ul) | 12 ul |
SpeI | 1 ul |
XbaI | 1 ul |
10xH buffer | 2 ul |
DW | 4 ul |
Total | 20 ul |
[[image:|thumb|pBad-RBS on pSB1A3 electrophoresis result]] [[image:|thumb|digestion result]]