Protocol Online logo
Top : Forum Archives: : Molecular Cloning

Screening PCR - DNA in wells? (Apr/20/2007 )


I just switch from miniprep to PCR to screen my colonies. I usually have two positive controls: a purified plasmid that contains a smaller part of the fragment I'm trying to clone (using the same primers as for the screening) and a plate of bacteria containig the TA-vector I cloned my fragment in before (using a different vector primer).
After the PCR I can see bands for both controls on the gel but no bands with the colonies I'm screening. However I do see some DNA stuck in the "screening" wells which isn't as bright as the actual control bands by far. I'm just wondering what that might be? Is this genomic bacteria DNA or is it possible that it is the fragment I'm looking for which (for what ever reason) didn't migrate on the gel?

Any idea?

Thanks! :-)


it is probably genomic DNA and other junk.

Do remember that colony PCR uses only a small quantity of cells. Too much cells and the PCR reaction fails.

I pick a middle size colony (about 1mm in diameter) and dilute that colony in 50ul sterile water (in 96 well plate). Using a multichannel pipette I take only 2.5ul of that 50ul colony solution, and add it to run my PCR reaction mix (total volume 10ul).