No band in PCR
Posted 02 January 2012 - 05:30 AM
Thanks for any help
Posted 02 January 2012 - 11:44 AM
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.
Edited by mahan, 02 January 2012 - 11:48 AM.
Posted 02 January 2012 - 12:17 PM
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?
- YanYan likes this
Posted 03 January 2012 - 12:31 AM
Edited by mahan, 03 January 2012 - 12:52 AM.