PCR techniques (help me) - (Apr/15/2005 )

Several weeks ago, I could do PCR very well. But i have no idea, why there days sometimes PCR works, sometimes not. I am sure i put the right component into the tube. Is there any important step people always will go wrong? did anyone have similar experience?

many thanks.

I made the experience that small amounts of ethanol inhibits the pcr reaction. for example if you clean your pipet with ethanol before pipetting the pcr reaction.

thanks very much.

I think maybe my problem is the ethanol. I did not clean the pipette by ethanol, but clean my bench by ethanol before my working. Maybe this is the problem. I will pay attention to this later.

I use 2 PCR blocks. both of them seems quite good before.

Thank you both of you for the useful idea.

Any other thing i should pay attention to?

i always vortex all the PCR components after thawing, before i add them to the master mix. maybe you can also try that
gr!

change the water! seriously, that's been overlooked a lot.