Team:HokkaidoU Japan/Notebook/aggregation Week 5

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Contents

July 30th

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.

July 31th

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

August 1st

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

DNA solution 4.5ul
SpeI 1ul
10xH buffer 1ul
DW 3.5ul
Total 10ul

SpeI 10xM

DNA solution 4.5ul
SpeI 1ul
10xM buffer 1ul
DW 3.5ul
Total 10ul


pT7-RBS(30ng/ul) =⑩

SpeI 10xH

DNA solution 3ul
SpeI 1ul
10xH buffer 1ul
DW 5ul
Total 10ul

SpeI 10xM

DNA solution 3ul
SpeI 1ul
10xM buffer 1ul
DW 5ul
Total 10ul

[[image:|thumb|digestion result]]

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

pT7-RBS(20ng/ul) =⑨ SpeI 10xH

DNA solution 4.5ul
SpeI 1ul
10xM buffer 2ul
DW 12.5ul
Total 20ul

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.

August 2nd

Preparing chemical competent cell

Preparing chemical competent cell of BL21.

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℃



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