Posted 01 March 2005 - 03:57 PM
I know what you're thinking ... this guy can't pipette, but I'm positive that I am getting template into every rxn tube. I even mix the template each time I add it to a tube.
I have replaced all of the reagents in the pcr reaction. MgCl2, 10 x buffer, and taq polm.
I do not believe that the problem is with my extractions; I have one assay that seems to be unaffected.
Posted 01 March 2005 - 08:46 PM
Hope this explains your problem too.
Edited by sharath, 01 March 2005 - 08:47 PM.
Posted 02 March 2005 - 04:00 AM
this possibility can be checked easily and quickly by running some test pcrs, containing all the same mastermix + template, but are run in differnt places of your cycler. If you see no difference in amplification afterwards, this possibilty can be ruled out.
Posted 02 March 2005 - 05:48 AM
Mike, I've had the same concern and will do an experiment, though I do have two thermocyclers and the problem has occured with both.
What about letting my template DNA get to dry after the 70% ETOH rinse before adding hydration buffer? This thought occured to me last night.
Posted 02 March 2005 - 06:06 AM
according to the fact all your samples should get the same conditions / time of drying, this is probably not the source of the problem... sorry but maybe you can let them dry whithout heating them (it changes result from my sequencing experiments regarding efficiency)...
if you think your P200 may be a problem, wash it good (sonication if a grat procedure for the plastic part of the pipette) and see if changes occurs.
Posted 04 March 2005 - 09:19 AM
in the past period ur lab has also had several PCR failures and guess what was theproblem,,,,thethermocycler weare using had little oil left in the test tube in the block
this caused differences between the block heat and the tube of the product temperature ....just adding little oil gave us good results
Posted 09 March 2005 - 05:58 AM
Thanks for the advice. I swithched from Taq to Amplitaq and my woes went away.