I can't find the DNA band of degenerate primer, I've done gradient PCR, DNA concentration variation, and touchdown PCR. The DNA I used is plant DNA. There is no problem with my DNA, because I have tried it with other primers as a positive control. how do I get a DNA band?
Should I make internal control markers for the degenerated primers? How to do it?
If other primer sets work then the problem is with this degenerate primer set. which may be old and degraded or possibly you are just annealing at too high a temperature. Calculate the melting temperature assuming any degenerate bases are A bases and set the annealing temperature at 5c below the lowest melting temperature
To obtain a degenerate DNA band using primers, you typically design primers that contain degenerate nucleotides at specific positions. Degenerate primers contain mixtures of nucleotides at certain positions, allowing for the amplification of DNA sequences with slight variations.
Here's a general approach to obtaining a degenerate DNA band using degenerate primers:
By designing and using degenerate primers in PCR, you can amplify DNA fragments that represent a range of related sequences with slight variations, allowing for the detection and analysis of diverse genetic variants or related sequences.
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