Methods for primer reconstitution - (May/03/2006 )
oh, I see; I'm sorry I was misinterpreting your difficulty
amine buffers (like Tris) cannot be directly treated with DEPC...that is why I suggested the alternate protocol...but it seems you are concerned about lack of pure water?
if you don't have access to milliQ or deI water, how will you set up your PCR once you have your primers taken care of? mdfenko's point (I think) is that if you have nuclease free water available to resuspend your primers, why can't you use it as the pure water you need to make TE?
I have promega's nuclease free water which is available is bottles in limited volume.My issue is basically contamination of water.
My question is:Is it safe for me to use autoclaved distilled or autoclaved DI water to prepare TE buffer? Does it ensure removal of nucleases?I don't think it will be safe for me to prepare my TE in just autoclaved water.
you should be able to make your TE in DI water and autoclave it
I regularly resuspend all my DNA in TE/DI water that has been autoclaved, usually there aren't any problems...DI water shouldn't have much anyways, as long as your talking about DNA work and not RNA...I think most DNAses are killed by autoclaving?
although I think many people use MilliQ water for 'extra insurance'
hmmmm but do u use also use that for making TE to resuspend ur primers?
Can u answer one more thing.Suppose i resuspend my DNA in TE and primers in nuclease free water will it have some negative effects upon my PCR results? 
hi
do as you wan, but if you can avoid TE and uses tris it's better.
A tip i do : when receiving new kits, the "elution buffer" is quite always 10mM Tris pH 8. So i aliquot it in clean tubes and uses it for tough material like primers.
do as you wan, but if you can avoid TE and uses tris it's better.
A tip i do : when receiving new kits, the "elution buffer" is quite always 10mM Tris pH 8. So i aliquot it in clean tubes and uses it for tough material like primers.
I was coming on this.I use TE buffer(tris-Cl pH7.4 EDTA pH8.0) to ehydrate my DNA.What say can I use this as well to resuspend primers?
Fred has a good point...you don't want a bunch of EDTA...let me clarify this for you; I suppose I have been lazy and perhaps not answering fully enough 
When my primers first come, lyophilized in their tubes, I resuspend them in TE, pH 7.6, at a very high concentration. I make small aliquots to a specific concentration and store them at -20C. When I want to make some for use, I thaw an aliquot and dilute the primers in either 10mM Tris (like what Fred has suggested) or nuclease-free water. You can use water here because the primers are already in solution, so you don't have to worry about that, see?
The issue of EDTA also applies with your template DNA as well, but I make a judgement based on what the final volume will be...for example, if I have a good chromosomal prep and I want to PCR a fragment, I will usually dilute the chromosomal prep to a workable volume (usually 100X and use 5ul/50ul reaction) with water or 10mM tris just prior to using as template. For example, if I thought I was going to be adding 3 or 4 ul of template to a 50ul PCR reaction, I would not use straight template diluted in TE because the EDTA will likely interfere with the PCR reaction. In this way, I suppose I am having to dilute more than some people will do, but the DNA as well as the primers seem to hold up in the freezer for a very long time with this system.
Call me foolish, but I reconstitute my primers in nuclease-free water.
I've never had a problem, nor should you. The truth is that a lot of things in molecular biology simply don't matter.
-Matt
plain, simple and cool water. Dont c why reconstitute in DEPC water.
I don't use DEPC water for my primers...I had misinterpreted and thought she wanted to make some nuclease-free TE...I was very confused... 