Creating short dsDNA from primers for cloning - (Aug/01/2007 )
Hello, I need help on the following subject:
I need to create a short (35 bp long) dsDNA oligo that is a certain region from some gene, and afterwards to clone it into double digested vector with compatible ends.
However, the problem is that one of the ssDNA primer that I need to anneal can create dimers, but I HAVE TO use it since it is a region from the gene.
My questions are:
1. how can I increase the specificity of the annealing in order to get rid of the dimer?
2. if I run it on a 4% sieving agarose, will I see the ratio of the dimerized primer vs. the correctly annealed primer.
The 5' oligo is 35 bp long and the 3' is 27 bp long and if they anneal correctly they create sticky ends on both sides.
well, anneal in STE buffer, standard way, and with 0,25, 0.5 and 1% DMSO.
You need those 3 tries to see the best which fit to your exp.
If 2 conditions are ok, use the less DMSO-containing-one for further exp.
For your gel, run in a lane the primer 1 alone, the 2 alone and then the anneal conditions.
You will see how behave the different partners.
Hi Fred_33, would you mind sharing the STEP buffer's recipie?