Hello, everyone
I've been struggling for almost a month already when amplifying a promoter from the whole mouse genome:
- I already designed 4 primers and have been trying them all (meaning a total of 4 pairs of primers)
- I've been adding 3% DMSO because it´s 60% GC rich and this has worked in the past with our PCRs
- Tried temperatures from 53-61ºC
- Every time I try a new temperature I try different magnesium concentrations (MgCl2+) starting from 0.6 microliters per 20 microliters of total volume until 2 microliters per 20 microliters of total volume (meaning from 1.5 mM until 5 mM)
- I´ve checked the DNA I´ve been using and it has an OK concentration (72.8 ng/uL) and 1.78 Abs 260/280
I really don´t know what else to try or check. I see no bands whatsoever and whenever I see something it´s only 200 pb and I am trying to get an almost 2k band.
Thank you very much in advance for all of your kind answers.
I will really appreciate it
Thanks again
what taq enzyme are you using?
do other primer sets work on your dna?
Are you seeing the size standard clearly on the agarose gel?
are you using primer3 or better for your primer design?
consider trying the pcr with a final concentration of betaine ( very cheap sigma sell It ) of 1 Molar
Let's see if we can rule out any sources of error.
Have you had success with other PCR reactions with the same reagents & equipment (enzyme, thermocycler, etc.)?
Do you have a good positive control DNA sample?
What are your cycling conditions? For a 2 kb product, you will want a longer extension step (at least 60 seconds).
What are you using for each reaction (primer concentrations & volumes, total reaction volume, etc)?
Thank you very much for your answers.
Is there any difference between DMSO and Betaine? I thought they were supposed to pretty much do the same thing and be good for high GC amplicons (although mine is not super high eiter, it's 60%).
I will try to answer them correctly with the information I have:
- No, I have not designed my primers with any program but they all match the basic criteria that we all know: roughly 50% GC, up to 30 pb (with the restriction enzyme site already added), tried it on Primer Blast before so I could see that it matches my desired amplicon... etc
- Yes, these reagents have worked fine with other PCRs very recently. I just succeded a week ago with the homologous human promoter and when I finally set up the right conditions I got a super clean and bright band, so I don't think PCR reagents are to blame.
- No, we don't have a positive DNA control since we are trying to get these promoters from scratch. Find the sequence, order the primers, use genome DNA...until we get it right, like I did with the human one (3% DMSO was the key). Very proud of this one when I got it :)
- Other primers sets work fine with our PCR reagents and mouse DNA genome (like, for example, when we analyze the genotype of our young mice using their fingers). So I don't think our genome DNA or the way we extract it from samples could be the problem...
For each 20 uL reaction we usually use:
2 uL Buffer 10X
2 uL dNTPs 10 mM
0.266 uL primer forward 20 uM
0.266 uL primer forward 20 uM
0.12 uL BioTaq Bioline
1 uL template DNA
0.6 uL DMSO (When I go for 3% DMSO)
H2OmQ + MgCl2 up to 20uL depending on chosen MgCl2 concentration
Also:
- I believe our extension time is 90 seconds
- 35 cycles
- Initial denaturing step is 3 min at 95ºC and we do that trick of heating it beforehand so that the samples go from ice to 95ºC immediately. Apparently it helps avoid primer dimers.
- Final extension time of 10min at 72ºC
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Sorry for the long answer, I tried to be specific and answer everything to make this easier.
Thank you so much!
Bea
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