I am trying to amplify pGBKT7 vector in DH5alpha E.coli. I made my own stock cell from an old stock in the lab. I did test my stocks for resistance by streaking on normal LB and on LB/Kan plates (50 ug/ml). They grew well on the LB and not at all on the LB/Kan plates. The problem is with the electrocompetent cells (prepared from my stocks by a lab-mate); after tranformations the control (no plasmid) grows a lawn on the LB/Kan plate and the 'transformed' cells do not contain plasmid. I have re-made the electrocompetent cells myself after retesting the stocks again twice (again did not show any resistance) and will try transforming again (fingers crossed).
Is it possible that the electrocompetent cells I used initially are contaminated, or could it just be a fluke that they all developed resistance suddenly?
Alanna James if the control (without any plasmid) is growing in selective media that means thr is 100% chance of contamination. Go for Cacl2- Dh5@ comp cells and heat shock transformation..
Hi Alanna James, You can check for contamination by plating out your electrocompetent cells (before doing the transformation) on LB+Km.
I have tried as suggested and there is definately something else in their stocks. Regardless though I have remade and am 100% sure mine are clean now. Thank you!
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