Primer concentration... - (Dec/19/2007 )
Hi all, i am new in this PCR primer stuff, i used to work with culture cells, but well now i am trying to do a PCR, i sent the primers to the core facility of my university and they gave me back a precipitate, with a note of 0.391 mgs, and all the protocols that i've read they work with different stock concentrations in uM how i know how much water do i need to add to this primer precipitate to get a 30uM conc. my primers are 30 bp
If anyone can help me i really appreciate, thank you
have you tried searching the archives for related posts?
concentration of primers in the reaction should be between 0.1 and 0.5 uM.
for the vast majority, i'd keep it to 0.2 uM.
when the conetration is too high, the higher the chance of mispriming.
look on the bottle, and see if you find the bit where it says the molecular weight.
here is a tool kit:
I have used primers at conc. between 0.2uM - 0.8 uM (final conc.).
I always prepare my primers at 10uM and use them at 0.1-0.5uM final conc.
here's a website to calculate the properties of any nucleic acid sequence.
I use Primers for Real time RTPCR, for that I prepare stock solution of 200 pM (forward and backward primers separately) and working concentration is 10 pM.
well if you have the mass of your primer, you should calculate the number of primer mole you have in your tube.
for this, i like biomath calculator, but there are others good too.
Then, if you have 300nmole, add 300µl and you'll have a 100µM stock solution.
Dilute a few to 10µM, to get the all-day stock solution.
work with 2 to 10µl of this solution in your pcr reactions.
note that some enzymes requires wery few amount of primer, and some others (like taq) can deal with primer concentrations ranging from 0.5 to 1 µM...