I am doing PCR with GJB2 primer regarding hearing loss. When i dilute the primer for the first time from the stock, it works only for 2 days. After it it does not work even i dilute it again from the stock. Can anyone help me what is the main cause of it?
I think it depends greatly on what you dilute your primers in. If you use water which has absorbed co2 from the air its pH is 5.5 so is acidic. The single stranded oligo will depurinate easily. This effect is more noticeable on highly purified oligos ( fplc or hplc purified). You may get away with adding much more primer as some may still be ok but oligos muist always be dissolved in alkaline solutions ideally with EDTA to remove magnesium and inactivate nucleases so use TE to dissolve your oligos. Also be careful storing your main stock. Some freezers are self defrosting and samples near the rear of these freezers can thaw and freeze often over the months and freeze/thaw will break up oligos
We use to face this problem when thawing the primers several times. As Paul said should check the solution the primers is diluted in. If the problem persist should consider the quality of your primers.
Good Job
- Badges
- Science method