annealing complementary oligos prior to biotin labeling - (Jun/18/2008 )
Hi all
i am trying to end label a pair of complementary oligos with biotin dCTP. I have a free G nucleotide at the 5' end of my reverse oligo. The process involves annealing the oligos first and then performing an extension reaction with the biotin dCTP. Usually when I check the labeling efficiency it is good but the set of oligos I am trying to label at present has a low labelling efficiency. I was wondering if this might be because the oligos have a high predicted Tm.
I've tried different annealing programmes
1. 950C 5 mins and then slowly cool to RT
2. 950C 5 mins, cool to melting temp of oligos and then hold at this temp for 30 mins before cooling to 40C
There wasnt much difference between the two programmes.
I've also heard that altering the salt concentration of the annealing buffer might help. Has anyone tried this and do you go up with the salt concentration to reduce specificity?
Many thanks!!!
i am trying to end label a pair of complementary oligos with biotin dCTP. I have a free G nucleotide at the 5' end of my reverse oligo. The process involves annealing the oligos first and then performing an extension reaction with the biotin dCTP. Usually when I check the labeling efficiency it is good but the set of oligos I am trying to label at present has a low labelling efficiency. I was wondering if this might be because the oligos have a high predicted Tm.
I've tried different annealing programmes
1. 950C 5 mins and then slowly cool to RT
2. 950C 5 mins, cool to melting temp of oligos and then hold at this temp for 30 mins before cooling to 40C
There wasnt much difference between the two programmes.
I've also heard that altering the salt concentration of the annealing buffer might help. Has anyone tried this and do you go up with the salt concentration to reduce specificity?
Many thanks!!!
I was having similar problems annealing my oligos until someone in the lab asked if I was using the right buffer. '"Right" buffer?' said I.
Here's what we use:
20 mM Tris, pH 7.4
10 mM MgCl2
50 mM NaCl
1 mM EDTA, pH 8
0.02% NaN3 (but I don't bother with this)
95 degreess for 5 minutes then cool slowly over a couple of hours. We usually boil about 500 ml, add the sample then let it cool to RT in the beaker.