Team:HokkaidoU Japan/Notebook/aggregation Week 7
From 2012.igem.org
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==August 16th== | ==August 16th== | ||
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[[image:|thumb|digestion result]] | [[image:|thumb|digestion result]] | ||
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+ | ==August 17th== | ||
+ | <div> | ||
+ | ==Colony PCR== | ||
+ | <p> | ||
+ | Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pBAD-RBS on pSB1A3 or not. | ||
+ | |||
+ | |||
+ | {|class="hokkaidou-table-pcr-reagent" | ||
+ | |- | ||
+ | |DNA solution | ||
+ | |4 ul (1 colony/10 ul DW) | ||
+ | |- | ||
+ | |Kapa-Taq(Taq polymerase) | ||
+ | |5 ul | ||
+ | |- | ||
+ | |Forward Primer(Ag43-f4 primer (50 pmol/ul)) | ||
+ | |0.5 ul | ||
+ | |- | ||
+ | |Reverse Primer(PS-R primer (50 pmol/ul)) | ||
+ | |0.5 ul | ||
+ | |- | ||
+ | |Total | ||
+ | |10 ul | ||
+ | |} | ||
+ | |||
+ | |||
+ | {|class="hokkaidou-table-pcr-time" | ||
+ | |- | ||
+ | |Number | ||
+ | |Degree | ||
+ | |Second | ||
+ | |- | ||
+ | |1 | ||
+ | |95 | ||
+ | |120 | ||
+ | |- | ||
+ | |2 | ||
+ | |95 | ||
+ | |30 | ||
+ | |- | ||
+ | |3 | ||
+ | |53 | ||
+ | |30 | ||
+ | |- | ||
+ | |4 | ||
+ | |72 | ||
+ | |60 | ||
+ | |- | ||
+ | |5 | ||
+ | |72 | ||
+ | |60 | ||
+ | |- | ||
+ | |6 | ||
+ | |4 | ||
+ | |HOLD | ||
+ | |} | ||
+ | Cycle:2~4 x 35 | ||
+ | |||
+ | We used N1 (DW only) and N2 (Ag43-dt on pSB1K3) as controls. | ||
+ | Desired product is about 800~1000bp. | ||
+ | |||
+ | [[image:|thumb|Colony PCR result]] | ||
+ | |||
+ | We thought that colonies of No. have band. | ||
+ | Next step, we resuspended colonies and cultured (add 1700 ul LB and 2 ul Amp) for hours in 37C. | ||
+ | |||
+ | </p> | ||
</div><div> | </div><div> | ||
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Revision as of 06:13, 17 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|digestion result]]
August 17th
Colony PCR
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pBAD-RBS on pSB1A3 or not.
DNA solution | 4 ul (1 colony/10 ul DW) |
Kapa-Taq(Taq polymerase) | 5 ul |
Forward Primer(Ag43-f4 primer (50 pmol/ul)) | 0.5 ul |
Reverse Primer(PS-R primer (50 pmol/ul)) | 0.5 ul |
Total | 10 ul |
Number | Degree | Second |
1 | 95 | 120 |
2 | 95 | 30 |
3 | 53 | 30 |
4 | 72 | 60 |
5 | 72 | 60 |
6 | 4 | HOLD |
Cycle:2~4 x 35
We used N1 (DW only) and N2 (Ag43-dt on pSB1K3) as controls. Desired product is about 800~1000bp.
[[image:|thumb|Colony PCR result]]
We thought that colonies of No. have band. Next step, we resuspended colonies and cultured (add 1700 ul LB and 2 ul Amp) for hours in 37C.