no PCR product - (Feb/26/2008 )

hi everyone! i'm very new in molecular biology. i need to do RAPD analysis on plant sample.i've done the extraction using CTAB method and my DNA template was quite okey.However i'm having problem with my RAPD. i couldnt get any band from my analysis

Here is my receipes :

o.125 microlit 0.5 microM primer (random primer)
1.5 microlit 3mM Mgcl2
0.1 microlit 0.02 U Taq DNA
2.5 microlit 1x Buffer
2.5 microlit 0.2mm dNTP
1 microlit 50ng DNA in 25 microlit reaction


My PCR condition:

94 C 3 min
94 C 45 s 30 cycles
37 C 1 min
72 C 1 min
72 C 7 min


i've tried it so many times but still no result.can anyone plz help me?i feel very depressed rite now with my PCR.thank you.

Am I getting this wrong?? Are you only using one primer??
You need to have a forward and a reverse primer for PCR....

Otherwise....
Do you have a positive control?? and if so, is that working??
Your annealing temperature seems awfully low, what is the Tm of your primers??

Hi there! Tahnx for your reply.

i'm just using one primer that is a random primer because i'm doing PCR-RAPD analysis.yes.i've have the positive control and same...no bands seems to appear on gel. I'm using 10-mer OPERON PRIMER with min Tm 39.5 and maximum is 43.6...what do u think?is it my PCR condition wrong? hurmmmm

Hi.

We are using the following Protocol for RAPDs. It uses another primer and was designed for fungi, but maybe some informations may help.....

http://www.scedosporium-ecmm.com/pdf/Fingerprinting.pdf

Are you sure that your template does not contain PCR inhibitors? As far as I know from our lab, a classical CTAB protocol may not remove all polyphenols etc. present in plant tissue.... so maybe you have to clean up your DNA?

hope this helps you!

gebirgsziege

I also get the feeling that your annealing temperature is quite low. Maybe you should try with something like 50 degrees. The primer could also be the problem; Are you only testing one primer, because in my previous lab they used to test several.

1.add more taq at least 0.2µL if the Taq is a hot start will need more denat time at least 5 min.
2. add 1µL of the primer
3. Tm too low calculate the Tm or use the one that comes in the primers document.
I think that the taq is stopping to work because the temp is too low.
try this:
95C 6 min 1X
94C 45 sec
?? C (calculate with 2(A+T) + 4(G+C) or use the provide by primer company) 1 min
72 C 1 min
Use at least 35 cycles
72C 10 min
10C

thank you for all the replies . currently i'm just using 1 primer (OPA-7) in order to make sure the primer is working.When everything is okey, i need to optimize about 50 primers but still i dont have any band on the gel at this moment .maybe the annealing T is so low.i'll try to optimize it..to gebirgsziege, thank you for that link..it was very useful...

This may sound silly but I spent 3 weeks getting no bands whatsoever from my PCR product. The problem? The electrophoresis tank is faulty. Found that out when even the DNA markers were not there. Some Taq manufacturer include the product sheet givin you the recommended PCR condition. It'll be good to follow then tweak it by yourself. Normally the annealing Temp begins at 50 - 68. Wish you luck.

Hello everyone..me again.Hurmmm...until today...it has been a month i'm working for PCR...but still no band appears.i've tried to manipulate every single parameter including by raising the annealing Temperature.But still no band i dont know what to do.for all my kind frendz...plzzz.....if u can help me..solve my problem...i would very much appreacite coz i'm very..very new in molecular....here..i enclosed my PCR product.hope i can get any suggestion for my failing result....thanx to all...

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If this is the first PCR reaction you have done, I'd recommend starting with something simpler. Find or make a simple set of primers and amplify a known template region of 400 bp or so, run it on a gel and debug your PCR skills and detection skills. This is not the place to start. I would also start (and likely stay) with a premix. This will dramatically reduce the problems with pipetting small volumes, which could be one of your problems.