presently I'm doing one study on ASSOCIATION OF COMT GENE VARIANT WITH ALCOHOL USE DISORDER.
the methodology consists of conventional pcr with comt primers and rflp with NIA III rest enzyme.
after conventional pcr I have got the 231bp gene of interest and usually after rflp if no mutation in the comt gene then there is no cut to the gene but if mutation is there, then 231bp gene is cut to 103bp,70,bp and 58bp.
but after following the NEB res enz protocol, there is neither non mutation band nor the mutation band is seen.
i have attached the protocols and gel pictures
It may be simply a matter of the amount of amplimer. When the enzyme cuts it produces shorter fragments that bind less EtBr so only fluoresce weakly so become less visible. Unless you are loading all of the rflp product it may be that your amplimer gets diluted in the rflp mixture and then produces smaller products that are faint and hard to see.
I would run more cycles of pcr (4 or 5 extra cycles). Check that your enzyme cuts in pcr buffer ( New england biolabs have a page for this) and cut most of your pcr product ( leave some for the uncut size control). Then do not run the gel too far as the lower distance it travels the stronger the band is.
Note This enzyme cuts well in ordinary taq buffer but not in high performance or proof reading polymerase buffers so is probably salt sensitive so you may need to purify the salts away and cut for longer but if using column purifications be aware that a lot of dna is retained on the column so you may need plenty to start with or use hot 80c elution buffer for twice as long as recommended by the column makes to elute your amplimer. You could consider small size exclusion columns to desalt your pcr product as the yields are better
Dear Anjan Rajkonwar ,
In RFLP if the PCR product is not in enough amount (concentration), you may lose the visibility with ETBr staining, this can be one scenario.
Secondly, the PCR amplicons may be destroyed by nuclease contamination as I can see in the Gel documentation that no bands were observed.
To overcome this, You can redo the experiment with more PCR amplicons (high concentration) and a fresh aliquot of Nuclease Free Water and Restriction Buffer and incubate the reaction as per the protocol which was 5-15 mins and instead of 50ul reaction, try with 30 ul reaction with 15-20ul of PCR amplicon.
I hope this will help you.
Thanks
Ananya
Before heading for restriction digestion of your PCR product you can do a purification and then start the restriction digestion. Run a standardization experiment of amount of PCR product and the restriction enzyme units. For visualization of the bands also run with a 50 BP ladder.
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