Hey guys,
I have a PCR product that is 66bp long and want to visualise in native PAGE but the bands are not as clear as I imagined they would be. I am preparing 10% gels that I pre-run for 30mins at 50V and then run the gel at 100V for 1h 15mins.
The DNA has a fluorescein 5' modification so there is no need to stain after running the gel. However, I don't have a fluorescent DNA ladder so running a bit in the dark (literally) in the PAGE gels.
Do I need to use even higher gel percentage or run it slower? Any ideas much appreciated
- I've included an example of what I see on an agarose gel vs the PAGE gels.
- The agarose sample is also on lane 4 of the first PAGE gel
- Last PAGE gel has PCR product and the ssDNA purified from that PCR
Hello Athanasios,
I am routinelly running 40 nt DNA oligos on 20 % PAGE without any problem and the bands are sharp and clearly visible. I suggest you to increase concentration of PA to 15 % (in your case of double stranded PCR product).
Hope this help
if your institute has a sequencer then linear polyacrylamide gels used in genotyping and sequencing capillaries are excellent at separating dye labelled DNA by both size or conformation depending on the temperature and denaturing properties of the POP gels. Other researchers doing genotyping studies will have size standards and you may be able to piggy back their runs if you do not have many samples at a time
Hey everyone,
I tried again with a 15% gel at 50V so they start separating evenly and after one hour I switched to 80V to speed things up. I also used a 66bp ssDNA I had ordered as a marker.
I'm surprised to see that all the PCR products seem to be further down than the marker yet all at the same length. And also the product from the last PCR I separated to ssDNA and purified and it also appears to be further up (same as marker). It's just more smeary.
I thought ssDNA runs faster than dsDNA but here it seems like the complete opposite is happening?
if you are running native PAGE then the dna may coil and form hairpins where there is homology. for dna to run accurately to size you are better using denaturing PAGE when all molecules are linear
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