Team:HokkaidoU Japan/Notebook/aggregation Week 5

digestion

I tried to digest the mini-prep products again, and after Ethanol precipitation, we did electrophoresis.

digestion result

transformation

I think that the E. coli which we use transformation is BL21(DE3)pLysS. So, the E. coli could multiplication increase on LBC plate.
I'm going to do transformation , use DH5α. Transformation of plasmid DNA ligated in 25th (pT7-RBS-Ag43-dT on pSB1K3) in E. coli(DH5α).

1. Added 2ul of plasmid DNA to 50ul of thawed competent cells on ice.
2. Incubated on ice for 30min.
3. Added 200ul of LB then incubated the cells for 2 hours at 37C.
4. Prepared and Labeled two petri dishes with LBK.
5. Plate 200ul of the transformation onto first dish and spread.
6. Added 450ul of LB to 50ul of the transformation and plated 200ul of it onto second dish and spread.
7. Incubated the plates at 37C for 14 hours.

liquid culture

Liquid culture for pT7-RBS-Ag43-dT on pSB1K3.

1. Added 2ml of LBK into culture tubes.
2. Resuspended colonies.
3. Incubated the tubes at 37C for

mini-prep

mini-prep of pT7-RBS-Ag43-dT which had been cultivated from yesterday. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50ul of DNA solutions. [[image:|thumb|mini-prep result]]

Digestion

pT7-RBS(20ng/ul) =⑨ SpeI 10xH

SpeI 10xM

pT7-RBS(30ng/ul) =⑩

SpeI 10xH

SpeI 10xM

[[image:|thumb|digestion result]]

We couldn't cut them exactry so we cut once more time.

pT7-RBS(20ng/ul) =⑨ SpeI 10xH

Liquid culture

Liquid culture for pBAD-RBS on pSB1K3.

1. Added 2ml of LBK into culture tube.
2. Scraped the surface of glycerol stock of construct.
3. Incubated the tube at 33C for OOhrs.