Real time PCR sudenly not working - (Jul/11/2012 )
Hello to all,
I am new to real time PCR, but I have done some successful experiments with Homemade protocol. I use AB7500 standart machine. A far as I know other people that use the same machine in the same time don t have problems. My problem is that one day I have normal Curves with Ct s around 28 and the next day I have no amplification at any well of my 96 well plate.
I have checked my aliquots (primers, buffer, enzyme, dNTPs, MgCl2 ) and they work OK.I can not imagine what goes wrong some times.
I would appreciate some help
Thanks.
-eldem-
eldem on Wed Jul 11 15:52:38 2012 said:
Hello to all,
I am new to real time PCR, but I have done some successful experiments with Homemade protocol. I use AB7500 standart machine. A far as I know other people that use the same machine in the same time don t have problems. My problem is that one day I have normal Curves with Ct s around 28 and the next day I have no amplification at any well of my 96 well plate.
I have checked my aliquots (primers, buffer, enzyme, dNTPs, MgCl2 ) and they work OK.I can not imagine what goes wrong some times.
I would appreciate some help
Thanks.
Hi,
Have you run the product which you got after your qRT-PCR. First run that in a gel which will confirm that you are getting product or not. Even I am using home made protocol for qRT PCR. Sometimes SYBR GREEN creates problem. Make a stock of 100X dye, whenever you want to use, dilute it and use it after use throw that away.
-ashu2007-