Re-suspending oligos in TE, Tris or water - (Oct/04/2005 )
I am wondering about resuspending oligos. I have always used TE for the 100 ul stock, and then diluted further with water. However, I am embarking on BAC sequencing, which uses primers at much higher concentration than my usual. Can I still get away with TE for the stock? Or will it cause problems? Anyone have experience with Tris buffer for this? I am reluctant to use straight water as the primers won't keep as well.
I have never used TE buffer for diltution of primers and used such primers for PCR, Primer extension and other molecular biology protocols. never had any problem. always used double autoclaved distill water to dilute the primers.
Yep -- me, too. Got to be a couple of thousand primers by now, all in sdH2O, no problems...
Most people use water and freeze at high concentration (100uM). Some companies recommend a TLE (tris-low-EDTA) buffer especially for resuspending taqman probes. Either one works fine, I think people just worry about the residual EDTA that may chelate the mg2+ in the reaction.
Years ago, companies that made your custom primers would not guarantee the primers activity if you dissolved in H2O. The amount of primer you received from the prep would last years and like all nucleic acids, some buffering is required to ensure stability. Therefore, you were told to use standard TE pH 7.5-8.0.
WHY - well, TE works better for nucleic acids compared to H2O when there is freeze-thawing involved. Most freezers used in labs were and still can be, frost-free, which means they go through a warm up cycle and thus your samples would defrost somewhat. Now, labs that can afford it, get the non-frost-free variety of freezers and avoid this problem.
I still resuspend my primers in TE at high primer concentration and then dilute in H2O for PCR, sequencing etc. Works everytime and I can still use primers resuspended from many years ago.
Hope this helps,