How to safeguard dNTPS? - (Dec/29/2005 )
After frequent failures of PCR reactions, finally we came to know that the culprit was the 20mM dNTPs which we were using these days. I wonder if any precautions to be taken for dNTPs similar to primers.
I don't know what others do, but I always aliquot mine right when they arrive.
like aimikins said, aliquot your stocks to reduce the number of freeze thaw cycles.
We have gone an extra step and made working stocks of dNTPs...........but nowadays for PCR we have PCR mastermix. Oh how those biotech companies make our life easier but at the same time takes a pint of blood off us.
Recently when we faced this problem with NO AMPLIFICATION IN PCR we also noticed that the pH of the MilliQ water was 5.3!!
Once again: use small aliquots, as frequent exposure to air changes pH of water, if I'm not mistaken by taking up CO2 from the air...
i also used to have problems with PCR and then we realized that its cos of freeze thawing of dNTPs.
making aliquots is the best way to preserve them. i usually make aliquots for 10 rxns.
at the same time, dNTPs from Roche r really good.....and quite stable, atleast it can withstand 10 cycles of freeze thawing for sure.
The pH of pure water (milliQ, RO, HPLC) is, almost by definition, nearly uncontrolled because it is not buffered. Very small amounts of any impurity will dramatically change the pH. The CO2 in dissolved air, in particular, will shift the pH to around 5. This is one reason pure water is a bad choice for dissolving oligos or storing DNA. The PCR buffer will easily bring the pH of the PCR reaction to the correct value, so the pH of the water is not important unless you are (mis)using it for dissolving DNA.
As EDTA is a known inhibitor in PCR I generally prefer using MilliQ for resuspending oligos, dNTPs etc.
just for comment :
i use aliquots of 10µl of dNTP and that's ok.
For oligos, 10mM Tris pH8 (the elution buffer of many kits) is ok and do not contain EDTA...
better to aliquote dNTP to avoid freezing and thawing