I have done PCR at different annealing temperatures and used Norgene Kit to extract DNA.
There is a just a band upper ladder in all of gels.
I checked my whole DNA (before PCR) by electrophoresis and there is a same band.
Does it means my PCR did not work? Why?
These are my primers:
Six 10-mer primers, Primer 1 (5’ GGTGCGGGAA 3’), Primer 2 (5’ GTTTCGCTCC 3’), Primer 3 (5’ GTAGACCCGT 3’), Primer 4 (5’ AAGAGCCCGT 3’), Primer 5 (5’ AACGCGCAAC 3’) and Primer 6 (5’ CCCGTCA GCA 3’) (Bangalore Genei, Bangalore, India)
Hi Reza,
How much template are you putting into your PCR? If you can see the genomic DNA band when you run the PCR reaction on a gel, that's too much. I usually use about 2-5 ng of DNA in a 50 µl reaction for most PCR.
Look Reza, could please upload you gel image. So that I can give better advice. In case if you can access old answers I gave replies reg. RAPD 3,4 times including my gel images.
In fact it is very sensitive PCR. Your hand should set for doing this kind of things. First of all I beleive your DNA is good. If that is fine entire thing work. Hardly 50-100 nano grams of DNA is sufficient per reaction as it is RAPD otherwise 10 ng of DNA is enough for normal PCR. May I know about your PCR content information I mean Mg Cl2, conc etc. and PCR profile.
And I would like to know what series of primers are you using. Generally there are hell lot of primers available, your choice, you can select any series, some give very good amplification with many bands minimum 10 or more some do not hardly 1 or 2, so you should try by taking 2 samples for screening of primers then you should select primers which give more band for your total number of samples.
Good Luck!!!
Hi!
As per the gel image your RAPD did not work, you too mentioned the same. Seems what ever the band seen is appearing as uncut DNA. By the way as per the publication did you digest DNA with different enzymes or not, check that. Even after digesting your DNA if you did not see any number of bands like in the publication your DNA is not at all digested, in the sense your REs (enzymes) are not working, are you incubating as per the protocol or not, for digestion even you keep longer times also it does not harm.
I believe you are extracting DNA with out any problem. But before doing RAPD you have to digest that with Hinf I, BsuRI, then you should see the same band pattern then go for RAPD, it should work and you should get the same band pattern with polymorphism..
Good Luck!!!!
Dear Jetty
I did extraction by Norgene kit (Canada). And also, I did RAPD-PCR no usual RAPD.
in any way, I agree with you. I think my extraction worked correctly but my PCR did not work.
Kind regards
It is fine but not mentioned whether you digested your DNA or not with those two enzymes HinfI and BsuRI. Whether it is RAPD or RAPD-PCR both are same, generally RAPD done at lower temp preferably 35C (Ta) by using single primer, all RAPD primers are decamers and that primer itself act as both forward and reverse, only thing these guys carried at 26C and 31C. May I know how do you differentiate RAPD and RAPD-PCR ?
I could able to see a band in your gel. Still you can try with some other primers.
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