Team:Evry/Notebook/Transposons/15.10.14

14.10.2014 ligation

it gave only one clone.

Clone culture:
5 ml LB + 5 ul Kan (25 mg/ml) - 37 C°

Transformation Pseudovibrio denitrificans & Dh5 alpha with mRFP in BBa_1413044

electrocompetent cells:

1 mm cuvette; 1800 V
recuperation media: MB for Pseudovibrio denitrificans , LB for DH5alpha

plating:

MB agar for Pseudovibrio denitrificans
LB agar for LB for DH5alpha

Transformation Pseudovibrio denitrificans & Dh5 alpha with BBa_1413001 in BBa_1413044

electrocompetent cells:

1 mm cuvette; 1800 V
recuperation media: MB for Pseudovibrio denitrificans , LB for DH5alpha


plating:

MB agar for Pseudovibrio denitrificans
LB agar for LB for DH5alpha

Verification testing of pnK2 in Pseudovibrio denitrificans

16 clones of Pseudovibrio denitrificans were grown in 3 ml MB, 30 C°, 24h

DNA extraction:

Invitrogen DNA extraction kit used with the Gram - protocol.
nanodrops gave value between 80 ng/ul & 130 ng/ul

PCR for Kan gene insertion:
PCR: enzyme: Q5; template: 200 ng genomic preparation Pseudovibrio denitrificans; oligo: F 82/ R 83; Tm tested: 55; elongation time: 2m00s

PCR for Pseudovibrio denitrificans specific oligo:
PCR: enzyme: Q5; template: 200 ng genomic preparation Pseudovibrio denitrificans; oligo: F 72/ R 73; Tm tested: 55; elongation time: 2m00s