Blah day in lab

[nwhiker]

Things did not work.

I'm annoyed because one experiment that I just cannot get to work is a colony PCR. Basically, set up a PCR normally, dip a pipette tip into a bacterial colony, and rinse it off in the PCR product.

I can NEVER get these to work. There is plenty of DNA in the reaction (it shows up in the wells of the gel, grr, where it does not belong), but the PCR part never works. I have much better luck doing an overnight culture from the colonies, isolating the plasmids of interest, and doing a PCR on them.

And yet my advisor keeps insisting on these stupid colony PCRs.

What's worse is because of enzyme issues, I had no positive control. Which means I don't know if the PCR itself failed vs the inserts all being in the reverse direction. I had ten colonies, I can't imagine that all ten ended up in reverse orientation so I'm blaming the stupid PCR itself.