Annealing the oligoes - (Aug/22/2005 )
Hi, do anyone know some simple way of confirming the annealing of oligoes. I am annealing two 64mers which I will be cloning in to retroviral vector. Am not getting the clone!. Was just wondering if problem is in the beginning itself!!
i'm havimg the same problem, when i run them on gel, ss Vs. ds do look different but not double bp position, which protocol u used to anneal, u might find prevoius discussion in this forum helpful, it even has a gel pic showing ss, ds bands.hope this helps
I've never really used a verification process for the annealing. If the cloning didn't work then I figured it was an annealing problem.
To anneal I would add the oligos together at some what equal dilute proportions in H2O or your primer buffer. Try a titration of each oligo as well if you want to cover all relevent concentrations for each. I've found that more dilute concentrations are more effective.
Using a thermal cycler or water bath, bring the oligo mixture to 95-100C for 5 min. Remove tube or multiwell plate from heat and let cool to room temp slowly.
This has worked great for me, but verify that your oligo construction is correct and and compatible with your vector and cloning strategy (sticky end, blunt, etc.).