I had run digestion of PCR products (366 bp) using RFLP. The expected products are 346 and 20 bp for normal, 366 bp for mutation. I already did the electrophoresis to check the digestion results with 3% agarose gel and ran at 60V for 1.5 hours but I still cannot differentiate between 366 and 346 bp band, both of them looked same. Here I attached the picture (the left lane is PCR product/uncut (366 bp), middle lane is digestion product (346 and 20 bp), the right lane is marker 25 bp). Could anyone give suggestion what is the best condition to differentiate those 366 and 346 bp fragments?
run a second/double digest restriction digest which has a common cut site in both amplimers as well as the one you are doing. Separating 150bp and 170 bp, for example, with a common band will separate much easier. Alternatively use different pcr primers to get a smaller product size after digestion as 20bp difference in smaller band sizes separate better
or try a gel at 4% because it'snt worth getting the 20bp band. the image is not very clear, is it right as what you get or a problem with the photography? if it's the way you see the gel, please use same buffer in gel and running buffer.
fred
Mr.Rutland: Thanks for your suggestion. Actually, I saw a 20 bp band in digestion product (middle lane) and no 20 bp band in uncut fragments (the left lane) as seen on the picture, I'm sorry for not so clear pic due to bad resolution camera. I will scan the result and show the real pict soon. There is a doubt, Is it okay if I only saw the 20 bp and ignored the differences 346 bp and 366 bp? I can't go back to change the primer due to limited sample volume and the work deadline.
Mr.Lepretre: Yes, there is 20 bp band on digestion product, Sir. I'm sorry for not so clear pic due to bad resolution camera. I used the same buffer (1x TBE) in making gel and running the gel. I will try using 4% agarose gel and report the results soon. Thank you for your suggestion.
Ok Novi,
if you run the gel slower and cooler ( cold room is ideal) but maybe at half your voltage for twice as long then the bands might diffuse less and be a bit clearer. I worry about just using the 20base band as in some amplifications it may be confused with a primer dimer but it does look quite convincing on your picture. If time were not a problem then you could have used PAGE which keeps the bands smaller ( less diffuse) . You could do a re amplification with just one new primer of the product that you have so long as you included the rflp in the new amplimer to get the total size shorter which would get round the small sample amount
If you are going to use the small band as diagnostic then do not run the gel as far as you are doing and the small band will be more intense and less diffuse. Maybe half as far as the picture you posted. Also run slowly as suggested before
Thanks for all suggestion. Finally 3% agarose gel with slow voltage and longer running time showed the 20bp differences of those 2 fragments.
good result Novi,
you could get a slightly stronger band using a comb with narrower teeth but that looks good. Thank you for letting us know
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