Basically, I'm trying to save time: as I'm doing qPCR almost on a daily basis, I am wondering if there is any reason not to prepare a master mix of H2O, forward primer and reverse primer in one tube (say, enough for a week or two of work) and keep it in -20C.
Apart from primers annealing to each other, I can't see any other potential problem in mixing them together. And since the first PCR step is denaturation, that should be irrelevant.
If there are no other problems with the method, the implication is that any primer pair could basically be mixed together and treated as only one PCR mix component.
Or am I overseeing some potential problem here?
It does work but I have one reservation. I do not like making up or storing oligos in water. Water in most labs has a pH of about 5 which is acidic and causes depurination so the oligos fall apart. Use alkaline TE or dilue pcr buffer. Generally also I do not like storing very dilute dna solutions ( or protein for that matter) as the plastic of some tubes adsorbs some of the oligo so while mixing is fine I prefer storage in a x10 mix. Primers are always in large excess so losing a little from a x10 mix is less important. Also I would not freeze the working stock of primers for 2 weeks...freeze thawing breaks oligos so I would store the mix at 4c if it is only for a couple of weeks
Hi there,
I've never used such premixes though I've been running thousands of PCRs... I don't think I've been wasting a lot of time...
But some have experienced these kind of premixes:
http://www.genengnews.com/gen-articles/7-tips-to-make-pcr-primers-last-longer-and-pcr-reactions-run/4150/
It does work but I have one reservation. I do not like making up or storing oligos in water. Water in most labs has a pH of about 5 which is acidic and causes depurination so the oligos fall apart. Use alkaline TE or dilue pcr buffer. Generally also I do not like storing very dilute dna solutions ( or protein for that matter) as the plastic of some tubes adsorbs some of the oligo so while mixing is fine I prefer storage in a x10 mix. Primers are always in large excess so losing a little from a x10 mix is less important. Also I would not freeze the working stock of primers for 2 weeks...freeze thawing breaks oligos so I would store the mix at 4c if it is only for a couple of weeks
Thank you all, especially Paul, for you answers.
The water I use is double distilled, so PH should be around 7. I understand that TE has advantages, but I rarely have problems with primer degradation, so ddH2O always seemed like a good-enough solution. In the past I only had problems when keeping primers at 4C for a month or so.
Thank you for pointing out the adsorption problem. My primer concentrations are near the upper end of the recommended range, so that shouldn't be a major problem, but I guess I could slightly increase them.
Thank you again for your recommendations!
Hello,
distilled water is acidic because it dissolves carbonic dioxide. So even if after distillation (which removes any gases) the pH is around 7, the pH will fall eventually.
I use a mix of two primers (25 µM each), no problems have ever occured, here's a citation from a user guide of the product: "Control primers are supplied as a mix of primers in H2O". I use them rarely, about once in a month, so they had about 15-20 freeze/thaw cycles already, still working perfectly (of course Phire pol is a big helper).
For more details please look at the step 7.1 in the manual (see the link).
https://tools.lifetechnologies.com/content/sfs/manuals/MAN0013358_Phire_Plant_Direct_PCR_UG.pdf
- Badges
- Science method