DNA enzyme digestion problems - Smeared and stuck in wells, no or faint bands (Dec/04/2008 )
Hi everyone,
I'm a research tech working with a mouse DNA strain that has been giving me problems for the last two months. In order to genotype this strain, we have to run a separate digestion after the PCR reaction using the enzyme BsteII to cut the DNA (otherwise, they all turn out nontransgenic). After digestion, however, the majority of the time it appears that the DNA is stuck in the wells (bright wells) and smears down the gel. While I know this usually indicates that there's too much template, I have varied the amount of PCR product added to this reaction.
The protocol I am currently using is from the lab that gave us these mice! For the PCR reaction, they use 37uL dH20, 5.17 uL buffer, 3.1uL 25mM MgCl2, 1.03uL 10mM dNTPs, 1uL of two primers, 0.4uL Bullseye Taq and 1.0uL DNA. For the digestion protcol, they use 3.0uL NEB Buffer 3 and 2.0uL BsteII enzyme added to the PCR product. THey then incubate at 60C for 1 hour.
Even though the template volume seems high, it works for them but it doesn't work for me!
OUr old protocol from Jackson Lab (7.96 dH20, 1.2 buffer, 0.72 MgCl2, 0.96 dNTPs, 0.3uL two primers and 0.06 taq with 0.5uL, 1.0ul, 2.0uL and 0.25uL DNA) + digestion (2.6uL dH20, 1.2uL NEB Buffer 3 and 0.2uL BsteII and 4-8.0uL PCR product) didn't work either. Though the 8.0uL PCR product digestion (compared to the 4.0uL) protocol worked temporarily.
I know it's not the PCR reaction because negative and and pre-digestion positive controls come out fine (though non-transgenic) with no smears or product in wells. The problem is after the digestion. I have varied the incubation time from 1 hour to overnight. The incubation temp is what is suggested for this enzyme. All primers, our enzyme, and reagents are newly ordered. Pipettes, dH20, and tubes are autoclaved. We use 1.5% agarose gel (though we've tried 1.0% to no avail) and run at 200V for 30 min (though I've tried 100V for 1hr.) We incubate in a dry incubator though I've used the PCR machine to incubate as well (when I thought evaporation might be a problem).
Any suggestions would be much appreciated. I'm out of ideas!
I'm a research tech working with a mouse DNA strain that has been giving me problems for the last two months. In order to genotype this strain, we have to run a separate digestion after the PCR reaction using the enzyme BsteII to cut the DNA (otherwise, they all turn out nontransgenic). After digestion, however, the majority of the time it appears that the DNA is stuck in the wells (bright wells) and smears down the gel. While I know this usually indicates that there's too much template, I have varied the amount of PCR product added to this reaction.
The protocol I am currently using is from the lab that gave us these mice! For the PCR reaction, they use 37uL dH20, 5.17 uL buffer, 3.1uL 25mM MgCl2, 1.03uL 10mM dNTPs, 1uL of two primers, 0.4uL Bullseye Taq and 1.0uL DNA. For the digestion protcol, they use 3.0uL NEB Buffer 3 and 2.0uL BsteII enzyme added to the PCR product. THey then incubate at 60C for 1 hour.
Even though the template volume seems high, it works for them but it doesn't work for me!
OUr old protocol from Jackson Lab (7.96 dH20, 1.2 buffer, 0.72 MgCl2, 0.96 dNTPs, 0.3uL two primers and 0.06 taq with 0.5uL, 1.0ul, 2.0uL and 0.25uL DNA) + digestion (2.6uL dH20, 1.2uL NEB Buffer 3 and 0.2uL BsteII and 4-8.0uL PCR product) didn't work either. Though the 8.0uL PCR product digestion (compared to the 4.0uL) protocol worked temporarily.
I know it's not the PCR reaction because negative and and pre-digestion positive controls come out fine (though non-transgenic) with no smears or product in wells. The problem is after the digestion. I have varied the incubation time from 1 hour to overnight. The incubation temp is what is suggested for this enzyme. All primers, our enzyme, and reagents are newly ordered. Pipettes, dH20, and tubes are autoclaved. We use 1.5% agarose gel (though we've tried 1.0% to no avail) and run at 200V for 30 min (though I've tried 100V for 1hr.) We incubate in a dry incubator though I've used the PCR machine to incubate as well (when I thought evaporation might be a problem).
Any suggestions would be much appreciated. I'm out of ideas!
As the DNA is stuck in the well, is it possible that 1 ul DNA that you use for PCR is very high concentration? For a genomic PCR, you don't need more than 500ng of DNA, even less would work. Just one suggestion.