2
$\begingroup$

I'm a little embarrassed to ask but when you have for example four degenerate primers and the end protocol says that the final primer concentration should be 10 µM working stock, should you make the primers 10µM total (2.5 µM each) or 10 µM for each degenerate primer?

$\endgroup$

1 Answer 1

2
$\begingroup$

You can just add 2µl from each working stock (10µM) to your PCR mix of 20µl.

You can check this protocol.
Caution: The author has used $l$ instead of $\mu l$

He uses 5µl of each primer (20µM) in a 50µl reaction. This is double of what I mentioned. IMO it is best to try with a lower concentration and then increase it if necessary. Excess primer concentration can give rise to some nonspecific binding and amplification if the primers are not perfectly designed.

From this site:

Using degenerate primers

Because a degenerate primer is really a mix of different primers, only a fraction of the degenerate 'primer' will actually work in the PCR. One would therefore need to increase the primer concentration in PCR, sequencing reactions, etc. If a primer is 8-fold degenerate, the concentration should be raised 4-8-fold, and so on. However, for higher degeneracies, e.g., 64-fold, one cannot increase the primer concentration 64-fold and would have to try their luck with a 5-10-fold increase only.

$\endgroup$

You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .