I am working on the resuspension of a fluorescent tagged oligonucelotide. I came across few protocols carrying out the process with either Tris-HCl or TE Buffer. The only difference in composition is the presence of EDTA. Would using either these two make a difference?
Prefer TE. EDTA chelates metals ions required by nucleases and hence increases the stability of the oligonucleotides.
Prefer TE. EDTA chelates metals ions required by nucleases and hence increases the stability of the oligonucleotides.
I agree with the answer given by Singh. Besides the stability, the effect of the elution buffer on downstream procedures should be under consideration. I often use a nuclease-free water instead of TE buffer to elute DNA for immediate downstream works. It works fine for me. In fact, I prefer TE buffer for long term storage of DNA
You may get amplification at 5 degree below the tm, but the optimum tm can be identified with a gradient PCR for maximum amplicon. For a first time PCR, I always perform a gradient PCR.
1mM EDTA used in TE for oligonucleotide resuspension (for 100 uM) would not affect PCR where the primers are diluted to almost 200 times (EDTA to 0.005 mM) and the necessary salts are several times higher in concentration. If or when its necessary to avoid EDTA, then use 10 mM Tris HCl with pH 8.5, at this pH most nucleases are least active, DNA is stable, and more soluble and therefore this buffer is provided for elution with silica column based DNA purification kits.
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