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.

Preparing chemical competent cell

Preparing chemical competent cell of BL21, JM109 and DH5α. Chemical competent cell made in each E.coli strains.

Our competent cell Protocol

1. Single colony isolation on LB plate
2. incubate the plate for 15-19 hours at 37℃
3. lift colony of E.coli into 2 ml LB
4. culture cells at 37℃ for 12-16 hours at 180-200 rpm
5. transfer 30 ul, 100 ul, 300 ul of the culture into 100 ml of SOB medium, respectively
6. culture cells at 20℃ (for 24 hours over) at 180-200 rpm (to ΔOD550nm=0.5～0.6)
7. leave the 300 ml flask for 10 min on ice
8. transfer the culture into two 50 ml Falcon tube
9. centrifuge 3000 rpm at 4℃ for 20 min (TOMY LX-120 rotor), and discard sup
10. suspend the pellet in ice-cold 15 ml of TB (Transformation Buffer)(7.5 ml/tube)
11. collect them in one tube
12. centrifuge 3000 rpm at 4℃ for 20 min (TOMY LX-120 rotor), and discard sup
13. suspend the pellet in ice-cold 3.2 ml of TB (Transformation Buffer)
14. Instil 0.24 ml of DMSO in precipitant
15. leave the 50 ml Falcon tube for 10 min on ice
16. divide 50ul of solutions in each 1.5 or 0.5 ml tubes
17. Freeze the suspension in liquid nitrogen
18. store at –80℃

Electrophoresis

Electrophoresis of digestion result of yesterday(pT7-RBS on pSB1K3 cut with SpeI)

digestion result

There were low concentration band in above of thin band. This thin band was same as digestion minus band.

Transformation

Transformation of pGEM into BL21. This transformation is the plastic production test in BL21.

1. Added 1ul of plasmid DNA to 50ul of thawed competent cells on ice.
2. Incubated on ice for 30min.
3. Added 600ul of LB.
4. Prepared and Labeled two petri dishes with LBA.
5. Plate 300ul of the transformation onto LBA dish and spread.
6. Added 900ul of LB to 100ul of the transformation and plated 300ul of it onto LBA dish then spread.
7. Incubated the plates at 37C for OOhrs.

Digestion

Digestion of pT7-RBS on pSB1K3(more fresh one)