MagA transformants grew on Ampicillin plates and Kanamycin plates but not Tetracyclin and Chloramphenicol
I0500 transformants grew on Kanamycin plates
pUC control also grew on Kanamycin plates and that's not good!
Suspected problems with preparation of our own Kanamycin plates and so re-do MagA, I0500 and pUC TOP10 transformants on Kanamycin plates prepared by Ellis
Plates incubated at 37°C overnight
Single Colony PCR of MagA from Ampicillin Plate
Without using master mix but normal protocol
Using updated PCR programme MAGA2
Touchdown: 6 cycles; Annealing max:56°C min:46°C
Normal: 28 cycles; annealing at 76°C
Reagents added in this order:
12.4μL SDW
4μL 5xHF Phusion Buffer
0.4μL dNTPs (10mM)
1μL primer MagAF (10mM)
1μL primer MagAR (10mM)
1μL DNA
0.2μL Phusion DNA polymerase
Run on 0.8% agarose gel with SyBr Green
Colony 4 has a faint band at 1.3kb for MagA
Quickly run colonies 1 and 5 on E-gel and re-confirm the absence of MagA in these 2 colonies but not the fault of agarose gel
Gel showing possible MagA PCR product at around 1.3kb from colony 4
Magnetic Bacteria
