We are performed an electrophoresis with primers taken from a paper based on hemiparasites samples. The posterior electrophoresis was different from the original work 160bp vs 700bp in several samples, can somebody have an explanation?
Many factors can influence the final resulta, Make sure first you re repeating the same conditions as published, (programm, final concentration of primers, MgCl2), your enzyme, Since you are obtaining a small fragments may be it's a degradation of your DNA sample that depend on the methos of extraction, purification and storage. or may be a non specific band du to the Low Tm , MgCl2 concentration.
In some cases your primers may anneal to unwanted DNA matrix, and the shorter product usually amplificates more effectively that the longer product. Check the primer pair using, for example, the Specificity Checking tool in NCBI Primer-BLAST. If primer anneals to matrix with 2 or even 3 mismatches, amplification is still possible.
Make sure that your samples aren't contaminated with bacterial or human DNA or free DNA sprinkled from other samples after PCR amplification. There may be also false priming sites in your target matrix, and primers may anneal to them with some mismatches if the PCR conditions aren't very strict (low annealing temperature, long annealing time).
Please read this article. This may help you...
http://www.readcube.com/articles/10.1038%2Fsrep05052
possibly the original paper worked on parasites in a different host and your host has some hology with your target dna .,Ithink the answer is primer specificity and the TM of primers depends greatly on salt concentration and primer concentrtion so maybe you are using a different enzyme buffer. I would either use DMSO or increase the annealing temperature in your pcr and, ideally, use a hot start enzyme to get rid of the smaller band which will often dominate the pcr because it melts easier than the longer band and amplifies more efficiently because it is shorter. Blast your primers against the host sequence or sequence the smaller band may give useful information
1-Checkout your DNA integrity before the experiment.2-Use a newly prepared working primer. 3-Re optimize annealing at a wide range of temperatures. Good luck
it is probably non specific binding. if problem is too severe then u can sequence the product and blast it and check it back.
- Badges
- Science method