No band in PCR - (Jan/02/2012 )
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
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.
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.
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.
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?
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.
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.