Another old blog article – this one written originally in July 2010 :
As PCR primers become less
expensive, is there less care being paid to them? Throw in a diluent,a quick
vortex and dilute for PCR?
I hope this doesn’t sound too familiar because even in these days of real-time qPCR and various probe chemistries, your PCR primers still reign king in the reaction. Your probe design is not going to determine the efficiency and – usually – the sensitivity of your PCR and qPCR reactions. Therefore its worth paying a little care in looking after your PCR primers – especially if they’re being used for a large number of runs and samples.
- upon receiving them, briefly centrifuge to bring the lyophilised contents to the bottom.
- To make up a stock concn of 100µM, take the number of nanomoles of primer delivered from the tube (check the tube) and shift the decimal point to the right, to get the number of µL to add. For example, from a recent design I ordered from Biosearch (whom we now distribute but I digress), I received 44.23 nmol of primer (50nmol synthesis scale). To make my stock solution, simply add 442 µL diluent.
- The best diluent? It seems to be a personal preference for everyone. I like to use TE’ (aka TE0.1) where the EDTA is at 1/10th the standard concentration normally in TE buffer. So that’s 10 mM Tris, 0.1 mM EDTA (pH 8).
- After adding the diluent, I leave the tube(s) on the bench
during coffeeah, for 10-15 minutes, a brief vortex, pulse centrifuge and I can make my working dilutions (typically 10µM).
Some suggest to make a more concentrated solution that can then be checked on a nanodrop (better: DeNovix ) to ensure accurate concentration determination but I’ve never had an issue with the amounts received (from Biosearch).
If you have some suggestions on looking after your primers then please add a comment or e-mail us