i isolated DNA from animal (gastropod), when i tried to amplify the DNA its only showing band from one sample only. I tried many times with many mixture composition but the only band i can get is from one sample and one species only, where may i gone wrong?
Total mixture i'm using is 20µL contain 200 dNTP, reaction buffer (pH 8.5), 1.5 MgCl2, DNA 20 ng, primer 0,75 µM.
When you use same master mix for both the samples, and if both of your DNA samples are good then you should get similar results in both. Let me know where is sample 2 in both the gels. In second gel, you can see primer dimers that suggests there in no amplification and PCR is not working. Your second DNA might have PCR inhibitors or the same second sample DNA was not properly solubilized. Or primers are not binding to template DNA.
In gel first in well no. 3 also you can see primer dimers.
i assume your DNA concentration is not 20ng, it must more higher, if it is 20ng you have to measure DNA perfectly...and use 10-50ng of DNA that might amplified all the samples.
HI,
From what I see on your gel is that you had (different) profiles for the majority of your samples belong different species, which is normal. The sample ho you had the maximum band must be the case that served to obtain PCR primers or close to it phylogenetically, other species should be further phylogenetically thereby either no amplification or the number of tape is reduced.
thanks for the answer... the first gel is actually one sample with different primer and the second one is different sample with one primer... i'll try to reduce the primer to prevent primer dimers and add more DNA...
It seems to me that some contamination is interfering with PCR. In the picture, it looks like the fragment amplification was quite limited. I would advise you to re-precipitate the DNA using ammonium or sodium acetate solution so you can purify the DNA a little bit and get rid of potential contaminants like proteins.
Semir
Since RAPDs are generated by random annealing using a 10-nucleotide primer, one could expect to have a number of fragments after each primer used. Keep in mind that these primers are unspecific and small so they can amplify at different places of the genome to generate a number of fragments. Depending on what you are comparing (more or less genetically related) the number of polymorphic bands will vary.
Adithya,
While working with RAPD primers, first step is to select optimal primers which are giving consistent band pattern in all the samples provided DNA concentrations are same. Pl test as many primers as possible.
Hi Adthiya,
There are several prerequisites and optimization steps before actual RAPD test to be performed which it should have done beforehand. i.e. DNA conc needs to be standardized across samples. PCR conditions such as conc of DNA, dNTP, primer, Mg and Taq should be optimized to generate RAPD profiles of high intensity with as many bands as possible and clear background. RAPD is also sensitive that sometime the age of DNA samples would cause failure in amplification. The longer you store, the more amplification problems will likely to happen. Hence good quality/high purity DNA would ensure better RAPD results.
Check the quality for amplification. Use any universal primer to test it with sample of DNA which is working and where it is not working. If You get amplification in both samples that proves DNA preparations are good else problem with DNA preparation. If DNA preparations are good then try to optimize concentration of dNTP, primers or template etc.
Good luck.
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