5 replies to this topic

[postdoc]

Hi All,

I am interested to know how you prepare your PCR stock solution to minimizing pipetting, do you put two primers in a single stock solution? Do you put all except primers in a stock solution?

Thanks.

[MoleculeMan]

I think what you can get away with depends on the intended use of your PCR product. I think if you are trying to get a pure amplicon without any primer dimers or anything, or if you are doing quantitative work, I would say that you should probably leave your primers separate and out of the mastermix until you are going to run. If PCR is for diagnostic purposes (which is what I do) and the only thing that you are interested in is the presence or abscence of a particular gene, then I would say go ahead and throw in everything and make your mastermix ahead of time. Although if your positive controls start to crap out, the first thing I would do is go back to the individual components.

I used to work in a place where the philosophy was everything had to be kept separate. Now where I work, they will throw all of their multiplexed primers into one tube (F&R) and save that as a working stock. I run so many PCRs that I am thinking about making large aloquots of complete mastermix (except Taq, call me superstitious) to save time down the road.

Edited by MoleculeMan, 29 March 2005 - 11:48 AM.

[AussieUSA]

Another alternative to the 4 suggestions in the poll is to mix everything (including primers) BUT the polymerase. Add the polymerase when ready to add to template and amplify.

I have been doing this for years - used to set up four PCR runs per day with 48 tubes per run (before the days of 96 well plates etc). I'd make multiple master mixes the night before and then on the day, add my template to the tubes, then the polymerase to the MM and then go from there. Worked beautifully

[lula]

hi

i agree with molecule man ...im currently working on stuff that requir the determination of the presenece or absenc of certain genes and to ave time and minimize pippetting errors i prepare master mix containning all the components includin the forward and reverse primers in the multiplex reaction im doing but i always leave the Taq OUT (I add Taq at the time of the run )

(i always get the feeling that the high glycerol in Taq requirs special respect )

[molgen]

For years I've been keeping the primers together in one tube and the mixes we buy have every thing in them including TAQ.
So, mix every thing up and don't worry about it.

[Wolverena]

postdoc, on Mar 29 2005, 02:27 PM, said:

Hi All,

I am interested to know how you prepare your PCR stock solution to minimizing pipetting, do you put two primers in a single stock solution? Do you put all except primers in a stock solution?

Thanks.

It mostly depends on the question asked / purpose of the PCR. If we just want to check the presence of a gene or if PCR is possible without using the PCR product for further downstream processes then we use already pre-made PCR master mixes or PCR beads where you just have to add your primers and template (and water if using beads). If I am using the PCR product for sequencing than I am using a proof-reading enzyme....in that case I am mixing the PCR solution with the individual ingredients when I need it. However, the primer sets we keep separate and only add them to the stock when they are needed, especially because we are working with several different primer sets (and sometimes mix and match different primers).