Making Primer Dimers on Purpose - odd I know...but humor me (May/30/2009 )
So I am trying to add a 3HA tag to the 3' end of my gene but I didn't want to use anybody else's HA construct b/c I needed unique restriction sites on both ends. My idea was to make primers that are complementary and each contain 1.5HA sequence, PCR them up without any template, and the primer dimer would be my product...my 3HA tag.
Any suggestions on my primer stock solution concentration...my normal -80oC primer conc. is 200uM.
This will work fine. The oligos should overlap by at least 15 bp or so at their 3' end. The primer concentration should be similar to that used in any PCR reaction. You should check the sequence -- oligos have a high error rate, especially long ones.
Good to hear. My overlap region is 25bp. The sequence and purity of the oligo was a concern but I am thinking that HPLC purification will be sufficient.
I would not bother with the cost of HPLC purification, but simply sequence a few clones after the fact. Remember to put sufficient 5' sequence to allow cutting with your restriction enzymes. I usually add around 6-8 bp.
Most people just design oligos containing your tag of interest (HA) and a sequence specific to your gene of interest. Use this in conventional PCR to amplify the tag and gene as one unit, then clone. I did this just a couple of weeks ago and it works fine.
Hmm...I didn't think to do it that way. There is more than one way to do anything so thank you for the different thought pattern.