Hello all, I have a query in resolving close products in agarose gel electrophoresis: I have three different expected products in my samples: 460bp, 480bp (only in mutation), 323bp, and I run them in a 3.2% TBE agarose gel, thickness 0.75cm, running conditions: 75V, at 4degreeC, for rough 8hrs with paused interruptions for detection every 2 hrs. What I see is that I have a definite signal at 323bp and 460 bp, then between 500-600 bps I have various other products (picture here after 8hr 10mins). I had cut the band, eluted the DNA and Sanger sequenced the products, turns out the band at 500bp is the same as 461bps and the band at 550bps is the same as the 323bps. The band at 460bps of the mutants, is a mixed signal in the middle of the Sanger sequence, where I could see that it has the sequence of the 323bps and the 480bps with the main 460bp sequence.
With the a second PCR of same settings and cdna, I run the sample with 3.2% agarose in TBE, reduced the thickness to 0.5cm this time, run it for 2 hrs in 150V, then further at 25V for 12hrs at room temperature. Here I do not see, multiple products between 500-600bps but one single product around 800bps. Can PCR products heteromerise when they run through a high % agarose gel?
I would like to resolve the products, but still avoid the poor resolution of some portion of the products, in the agarose gel. I am using the biozym LE agarose. any suggestions to improve for this experiment please?
you would get better separation if the pcr products were smaller. Can you find a restriction site in both N and M samples so that you are separating, for instance, 120 from 140 or even redesign your pcr primers so that the pcr product is only 120 bp
Paul Rutland thanks a lot for your suggestion, unfortunately I can not go down more than 370bps, because of the size of my insert DNA. do you know any suggestions for other methods for a higher resolution of close bands?
Linear acrylamide in capillary electrophoresis will separate well but is not useful preparitively or just long PAGE gels if you just want to see band separation
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