Annealing and labeling oligo probe for gel-shift assay - (Oct/11/2005 )
I have a problem of making DNA probe for gel-shift assay. I made the DNA probe by annealing two 24nt oligonucleotides at 65oC for 10 mins followed by 32P labeling, but the radioactivity was much lower than the normal one when I checked the quantity of radio-label. Since I had good result for my positive control, so I guess there may be some problem of primer annealing. Because my DNA primers are not good enough that it has some probability of self-annealing and mismatch, so I am wondering how can I improve its annealing, increase the temperature, and how high is it if I have to use this pair of primers? Thanks a lot.
For annealing oligos, I heat oligos in annealing buffer at 94C in a beaker with water and let the water slowly cool down over one hrs period to 37C. After that, I run a gel with ss-oligos to check if ds-oligo has been formed or not. Also oligo quality is very important for such procedure.
Check this page for oligo annealing protocols
Hope that helps.