I am doing PCR-RFLP to detect SNP, after the restriction enzyme digestion I check with 15% polyacrylamide gel, if the restriction occurs I should see two bands (126 and 20bp) but I only see the 126bp band, what could I be doing wrong or how do I solve it?
A short 20 mer traps very little intercalating dye so is diffuse due to its short size and weak. You might see it better by running the gel for a shorter time so maximising the signal and using silver staining to detect the bands but why do you need to see the 20mer. If you run an uncut 146 size amplimer then any sample generating a 126 base product must have been cut by the enzyme and 146 and 126 will separate well on a wide range of PAGE gels
20bp is a very small product. If you have any primer-dimers, the 20bp will be hidden by that "smear" on the gel. And unless your starting products are extremely bright, the 20bp just won't give off enough brightness to be visible.
Paul Rutland 's suggestion of including an undigested 146 bp product as a comparison will help you interpret your results.
There could be a few possible reasons why you are not seeing the 20bp band in your native polyacrylamide gel:
If you have tried adjusting the gel conditions and confirmed that the restriction enzyme is working properly, you may need to try optimizing your PCR amplification or DNA template preparation to improve the yield of the 20bp band.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
- Badges
- Science method