Primer Dilution. ...water or buffer? - (Jun/01/2007 )
Hi guys...
I am currently using 10X PCR buffer containg MgCl2...I did not used this buffer to dilute my primers but water. To avoid unecessory calculation (for Mg and others) in preparing master mix. My friend is not happy on this...of course my primers are working wel.
What do you advice me?
Txs
No water no life.. water is your best friend, crystal as water. let the water run.. no prob (as long as its milliq)
I am currently using 10X PCR buffer containg MgCl2...I did not used this buffer to dilute my primers but water. To avoid unecessory calculation (for Mg and others) in preparing master mix. My friend is not happy on this...of course my primers are working wel.
What do you advice me?
Txs
I agree. There is absolutely no problem with diluting your primers in water, I have never heard of anyone diluting them in buffer.
I dilute my primers in 10mM Tris, pH8 (some use 7,5) buffer. It's actually only water buffered to a certain pH. This should prevent degrading of the DNA in lower pHs.
We elute all our DNAs in buffered water.
we use milliq to dilute primer. if you are sensetive then you can use milliq from just autoclaved bottle (ofcourse after cooling)
I use TE. The EDTA prevents most DNA enzymes from being active, making the DNA much more stable. The small amounts of EDTA added to a PCR reaction with the very small percentage of the mixture coming from the primers is negligible: TE has 1 mM EDTA, and (at 30 pmol/ul) will be a maximum (with both primers) of 3% of the PCR volume. This means it will chelate 0.03 mM of Mg++ from the reaction, out of typically 1.5 -2 mM Mg++, a totally negligible amount.
we use millipore water to make primer solutions. They are quite stable. In my old lab we used TE. So both of them work beautifully.
Used Nuclease Free Water that comes in the DNA Purification Kit. Is that in anyway diff from MQ?
The kit water may be your best bet since that stuff is QC'd before packaging with kits. MilliQ or nanopure water can also be fine, but I'm a fan of autoclaving, since you never know where your glassware has been. I usually use TE as well for extra insurance against degradation/contamination.
I've used both water and TE. At first I was concerned that EDTA would chelate Mg++, and reduce the activity of Taq, but actually it works fine. Probably the EDTA concentration is not high enough for that, but it still protects my primers. So I just use TE from now on.