Hi everyone,
I have a couple of high GC content oligonucleotides that I need to anneal each other. One of those is 6-Fam labeled. For me it is important to have all the 6-Fam primer annealed. For this reason I use 1:2 ratio between non-Fam and Fam labeled primers during the annealing, but I can get only 80% efficiency.
My annealing buffer is 30mM HEPES pH8, 100mM KCl, 2mM MgCl2, 50mM NH4Ac.
For the annealing, I heat the oligos at 95°C for 5 min. Then I shut off the heater and let the temperature drop to room temperature.
Thank you for any help.
if you want to shift the equilibrium to use all of the fam primer use a large excess of non labelled primer (10-100 x) and leave the annealing stage longer 95 seems unnecessarily hot ...the oligos are already single stranded so you are only straightening out any self annealing so 80c for a few seconds should be enough. Check the oligo sequence of your fam oligo for hairpin self annealing and if this is possible hold the annealing temperature at a temperature where self annealing cannot take place but oligo-oligo annealing is possible....something like 80c for 30secs then 50c for 4 hours as an example. possibly you can get rid of the excess unlabelled primer with EXO1
Hi Paul, thank you for the suggestions.
Indeed, my Fam oligo forms several hairpins (I design it in this way on purpose). The predicted Tm of those secondary structures are around 43°C. As you suggest I'm trying 1:20 ratio and I'll also add few long steps with temperature above self-hairpin Tm (60° and 50°C).
I'll let you know how it goes, thanks again,
Emmanuele
Edit: It worked perfectly!
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