Probably it's a stupid suggestion, but did you include a positive control to make sure your HindIII was cutting properly?! Otherwise, can you exclude the possibility that there are indeed no polymorphisms? You could give your PCR fragments for sequencing to be sure.

Best,

Cindy

Probably it's a stupid suggestion, but did you include a positive control to make sure your HindIII was cutting properly?! Otherwise, can you exclude the possibility that there are indeed no polymorphisms? You could give your PCR fragments for sequencing to be sure.

Best,

Cindy

If hind III is not showing polymorphism then try to digest with some other enzymes both to reference and test sample pcr amplicons. If  not then go for sequencing if it is feasible option for you or else if the amplicon size is upto 600 bp or below then do denaturing gradient gel electrophoresis to see any difference in banding pattern in test sample compared to reference amplicon.

Good luck

The best way is to have a positive control, in RFLP digestion, which will confirm that the enymes are fine. You can randomly sequence some of your samples to confirm that there is no restriction site in your sample sequence for HindIII.. And also where do u get your enzymes (NEB/Fermenotos/any other source). Check it with a positive control first, i mean a definite sequence having the restriction site for HindIII. Good luck hope it helps

Dear Hasan,

First digest 1ug of Lambda DNA with your HindIII enzyme, with 1X final buffer concentration at 37 deg C for 1 hour and then check on 1% agarose gel. You should able to see minimum of 5 fragments having one at around 27Kb. If you are using fast digest then you can adjust the time. This will help you in identifying whether the enzyme is working or not.

Secondly, go for DNA sequencing of your PCR product, then check for any loss or gain of restriction sites for HindIII using Restrictionmapper tool. If you get, restriction site for the HindIII then you can digest, but if there is any loss of restriction site, then you may not find any digested fragments.

All the best

Rajkumar