Inconsistent colony PCR result (View forum version)



Lian

Posted 15 December 2014 - 09:08 PM

Hi, 
I am doing bac to bac transformation of DH10Bac. 

Blue white screening was used to select the transformed colony.

White colony was picked and used for colony pcr and re-streak on new selective plate. 

The PCR result showed no amplification at all, while the colony re-streak on new selective plate still white. 

The transformation protocol, competent cell, and PCR reagent and protocol were same batch with my previous experiment which I manage to amplify my plasmid. 

 I did tried to purify the plasmid and analyze using gel electrophoresis, and this gave me positive result, however, the extracted plasmid still can't be amplified by PCR reaction.   

* The condition of all PCR reagents had been tested using my lab mates samples, and their samples can be amplified (but with faint band). 

Thank you. 


bob1

Posted 15 December 2014 - 09:42 PM

Colony PCR often runs into the problem of too much DNA and inhibitors produced by the breakdown of the bacterial wall. Try dipping the picked colony into 100 ul of water and then using 1 ul in the PCR.


Lian

Posted 16 December 2014 - 12:47 AM

Hi Bob, I had tried this before, but still showed negative result.