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Team:HokkaidoU Japan/Notebook/aggregation Week 7
From 2012.igem.org
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In this result, N2 has about 500bp band, but N1 has not it. | In this result, N2 has about 500bp band, but N1 has not it. | ||
| - | So, we use to liquid culture, No. 7,10,12. | + | So, we use to liquid culture, No. 7,10,12.<br /> |
| - | + | Next step, we resuspended these 5 colonies and cultured (add 1700 ul LB and 2 ul Amp) for 15 hours in 37C. | |
</p> | </p> | ||
</div><div> | </div><div> | ||
Revision as of 07:52, 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 |
Gel extraction
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
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.
In this result, N2 has about 500bp band, but N1 has not it.
So, we use to liquid culture, No. 7,10,12.
Next step, we resuspended these 5 colonies and cultured (add 1700 ul LB and 2 ul Amp) for 15 hours in 37C.
