I need to get one single band from my sample to sequence the target . what should I change?
I changed annealing and DNA concentration, time of each cycle and used different PCR master mixes.
Your sample may be contaminated. Could you provide a picture of your results, and which protocol you used to get your sample?
I have no idea what kind of sample you did your PCR on. It might help to provide more detail.
But this way might help:
Try PCR a larger size, which includes your 450bp first, purify the product, and use this fragment to amply your 450bp DNA fragment.
If you still cannot get a larger PCR product with a new primer set, there may be something wrong with your sample.
It may be that your sample has a polymorphism near the 3' end of one primer so I think Adam Lin is right. Amplify a larger fragment and sequence it to make sure that your primers (internal) can anneal and then run the nested pcr
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning