Team:HokkaidoU Japan/Notebook/aggregation Week 7
From 2012.igem.org
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 |
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.