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No band in PCR


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5 replies to this topic

#1 mahan

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Posted 02 January 2012 - 05:30 AM

I setted up my pcr last week. I did it for 20 samples. but it does not work. and I dont know its reason.
Thanks for any help

#2 phage434

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Posted 02 January 2012 - 05:53 AM

I went for a walk, and got lost. Thank you for any help.
Seriously, what can you possibly expect as an answer? We know nothing about what you did or want to do.

#3 mahan

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Posted 02 January 2012 - 11:44 AM

I study some related genes in heart disease by RFLP- Based PCR. After getting samples from heart patients and dna extraction making by Qiagen kits, I could sett up pcr reaction by this program:
Initial denaturation: 95 for 1 min
Number cycles: 40
Secondary den: 95 for 1 min.
Annealing temp.: 54.4 for 1 min.
Extension temp.: 72 for 1 min.
Final Extension: 72 for 10 min.
and this:
10X pcr buffer: 2.5 microL
50mM Mgcl2: 0.75 micL
10mM dntp: 0.5 micL
R and F primers: each 2 micL
Taq pol: 0.25 micL
Template and ddH2o: 1to 17 micL

. first, the reaction efficiency was well and It had very good bands,But now it doesnt work and I have no band while all terms are as before and I dont know what thing has changed.
Thanks

Edited by mahan, 02 January 2012 - 11:48 AM.


#4 phage434

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Posted 02 January 2012 - 12:17 PM

How is your DNA stored? How much DNA are you adding? Do you have a control reaction that you can run?
Some guesses: perhaps you are using too much template DNA, which can inhibit a PCR reaction. Try 10x and 100x dilutions.
Perhaps your reagents are bad. The dNTPs, in particular, are sensitve to freeze/thaw. Perhaps you need to adjust the annealing temperature down a bit. Are you sure it is the PCR that is failing, rather than your gel? Does the ladder look good on your gel?

#5 mahan

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Posted 03 January 2012 - 12:31 AM

Templates extracted from deifferent samples and then loaded on 1% agarose gel. Therefore according to their concentration on the gel, appropriate amount of template ( 1 to 17 micl)is added to the reaction . After extraction, templates stored in the freezer(- 4 centigrade). I think gel works well. I try it and ladder is good on gel. dNTP is new. Unfortunately I havent control reaction. Mgcl2 and buffer checked by others.

Edited by mahan, 03 January 2012 - 12:52 AM.


#6 phage434

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Posted 03 January 2012 - 05:57 AM

I'd encourage you to try diluting your template DNA. You need only a ng or less, and large volumes as a fraction of the reaction can allow inhibitors from the gel extraction to cause problems. Most of your pcr volume should be water.




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