Team:Evry/Notebook/Transposons/16.10.14

Trying to show BBa_1413044 work even empty in DH5alpha

8 colonies of DH5alpha were transformed with BBa_1413044 and grown in 3 ml LB + 2 negative control

Preparation for PCR colony:
Spin down 500 ul culture at 14000 g - 2 min, resuspend in 100 ul H20 MilliQ, put at 95 C° for 10 min then spin down at 14000 g - 2 min.

PCR: enzyme: Q5; template: 3 ul of supernatent for the culture; oligo: F 100/ R 101; Tm tested: 55; elongation time: 1m00s

The two last wells are the controls which surprisingly displayed a band, and we did not obtain a single band for the 8 strains. In the end the gel revealed itself to be inconclusive with regards an empty transposon vector.

defining where the transposase integrate

find enzyme with a good cut numbers in the genome and not in our insert: HindIII Digestion of 8 Pseudovibrio denitrificans DNA preparation by HindIII
50ul final volume, 200 ng DNA preparation, 5 ul Neb 2.1 buffer, 38 ul H2O, 1 ul HindIII. 1h

Then religate:
Ligation: 20 ul final volume, 2 ul 10x T4 ligase buffer, 1 ul T4

then light digestion with XbaI
50ul final volume, 20 ul DNA religated preparation, 5 ul Neb 2.1 buffer, 38 ul H2O, 1 ul XbaI enzyme. 15 min
deactivation at 80 C° for 20 min

then PCR
PCR: enzyme: Q5; template: 3 ul of digestion by XbaI; oligo: F 105/ R 106; Tm tested: 55; elongation time: 4m00s

Oct 16