Team:HokkaidoU Japan/Notebook/Week 3

Ag43, dT

Digestion

Results of digestion in 15th.

Digestion result image

Product:Ag43(K346007)=3120bp and 2070bp, pT7-RBS on pSB1K3=2247bp We confirmed there are some kind of restriction enzyme site in K346007 (digested with SpeI, PstI) and pT7-RBS on pSB1K3 was successfully digested with EcoRI and PstI.　Balance between d+(EcoRI) and d+(E&P:cut with EcoRI and PstI) is about 80bp.

Gel Extraction

Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics). Got 50ul of DNA solution.

Ethanol Precipitation

Ethanol Precipitation for digestion and gel extraction products.

1. Added 5ul of NaoAc, 1.5ul of glycogen and 125ul of 100% ethanol.
2. Centrifuged in 15000rpm, 10min at 4C.
3. Remove supernatant and added 220ul of 70% ethanol.
4. Centrifuged in 15000rpm, 10min at 4C.
5. Remove supernatant and air drying in room temperature then added 5ul of DW.

dT(B0015) would be amplified　incorrectly. So we tried another DNA amplification method: PCR.

PCR

PCR for dT(B0015)

Cycle:2~4 x 45

PCR result image

We migrated B0015 mini-prep psoduct, digestion product, and PCR product. PCRed dT would have 429bp(100bp(added by forward primer) + 129bp(dT) + 200bp(added by reverse primer)). Most bright and thick band in this image has about 300~400 bp. We thought dT was amplified successfully.

Ag43

Digestion result of Ag43 was incorrect. We digested Ag43 once more time.

Digestion

Digestion for Ag43 with SpeI and PstI.

[[image:|thumb|Digestion result image]]

Liquid Culture

Liquid culture for single colony isolated pT7-RBS on pSB1C3 and Ag43-dT on pSB1T3.

1. Picked up one colony from single colony isolated plates　by platinum loop.
2. Dipped into 2ml of LBC and LBT.
3. Cultivated.