currently, I am working in PCR-RFLP. I just get my PCR result sequenced, and the result shows that my PCR sequence has the restriction site. But, in electrophoresis agarose, the digested DNA band showed it was not digested.
I have already incubated the digestion process for overnight to make sure that the enzyme work.
what should I do? what do you think is wrong? I've already checked the restriction enzyme in NEBcutter, and it was the right enzyme for my amplicon. Thank you for your answer.
Hi,
try to run positive control to make sure, your enzyme works under given conditions.
Most restrictases (DraIII especially) do not like PCR buffer. It is recommended, that the volume of restriction buffer is decreased by 30% when cleaving eg 5-10 ul of PCR product in the final volume of 15-20 ul. You have 20 ul of PCR product in final 25 ul reaction, what seems to me quite a lot.
You can either dilute your restriction at least to 50 ul (use 20 ul af amplicon 3.5 ul of buffer (10x), 1ul of Dra III, and water to 50 ul), or desalt your PCR product (EtOH precipitation, Sephadex purification,...) before digestion.
Good luck Martin
Can you provide more information like your target gene, which enzymes you are using and at what conditions? This will help us answer better
I use mutagenic primer which make restriction site TTT | AAA, and Dra I as the restriction Enzyme. For digestion mix, I used 20 microliter of amplicon, 2,5 micro of Buffer, 1,5 micro of ddH2O, and 1 micro of enzyme. It is incubated in 37 C overnight. If it is digested, the size of the band should be around 120 bp (the amplicon size is around 140 bp)
The digestion result band, didn't show the size of 120 bp, but likely same with the amplicon size. But, in sequencing result, there is the TTT | AAA site. So, what could possibly wrong? And what should I do next? thank you for your help :)
Hi,
try to run positive control to make sure, your enzyme works under given conditions.
Most restrictases (DraIII especially) do not like PCR buffer. It is recommended, that the volume of restriction buffer is decreased by 30% when cleaving eg 5-10 ul of PCR product in the final volume of 15-20 ul. You have 20 ul of PCR product in final 25 ul reaction, what seems to me quite a lot.
You can either dilute your restriction at least to 50 ul (use 20 ul af amplicon 3.5 ul of buffer (10x), 1ul of Dra III, and water to 50 ul), or desalt your PCR product (EtOH precipitation, Sephadex purification,...) before digestion.
Good luck Martin
Martin has given the most important recommendations especially running a positive control to ensure that the enzyme is actually working. That will be a good place to start
I agree with Martin, you can reduce the amplicon volume as well. Try to incubate for one hour ,which is enough , as longer incubation time might degrade small DNA fragments and give you a smear on electrophoresis or invisible bands. Then run the original sample in an agarose gel of 3% . Adjust your time as well. Good luck
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