water!!!

For the past two weeks, I've been working on amplifying a gene encoding three proteins fused together - yellow fluorescent protein:beta-galactosidase:cyan fluorescent protein - by PCR. Unfortunately, all I've been able to get were empty gels when I ran the PCR product on the gel. I've manipulated several conditions and components of the reaction and still came up with nothing.

Yesterday, Dr. R suggested that I try using the DNA Grade water from Fisher for the reaction or get a new batch of dNTP mix from Invitrogen. I did both but to amplify the 16S rDNA gene from Verrucomicrobium, which is a positive control and guess what..the reaction with the DNA Grade water worked!!!!

Just before I left work today, I started the reaction to amplify the yfp:lac:cyp gene, which was my initial objective. Will only get to see if it works tomorrow but I have a good feeling that it'll work. Can you believe that something as simple as water can screw up experiments for two weeks??? I autoclaved my water for 30 minutes and aliquoted it out into sterilized Eppendorf tubes and never did open it again until I need to use it. So the only possibility that's screwing up the PCR is probably some mineral residues in the bottle during washing or there is an inhibitor in the bottle and it was not washed thoroughly.

I can't wait till tomorrow to check it but I'm soooooo glad I find out what's wrong with the reaction for positive control.

~ecstatic~