IQ5 and sybr/FAM (View forum version)



peanuts

Posted 02 March 2010 - 09:16 PM

I am new to Q-PCR, and have problems with the technical replicates. I run triplicates for each gene, and the CT varies significantly between the replicates (~2). It is quite unlikely that this is due to pipetting error, since the CT difference is so high. Then I try to use "persistent well factors" and it seems to improve the variance a lot.

I wonder when I use "dynamic well factor" for cybr green/fluorescein, do I need to setup anything so that the machine can detect fluorescein? Since the manual says it's a automatic process, I suppose I do not need to do anything when I use "dynamic well factor" (calibration)?

I appreciate if anyone can save my life........

dreww

Posted 18 March 2010 - 05:28 PM

You don't need to. Just do a persistent well factor and a pure dye calibration

peanuts

Posted 19 March 2010 - 10:30 AM

Thanks for your reply. I also found that persistent well factors work better for me.

dreww

Posted 20 March 2010 - 01:49 AM

Sounds good. the smallest difference in primer concentration/amount, polymerase, template, etc. makes the biggest difference in your ct value. try your best on your pipetting and make master mixes of your replicates. replicates are there for a reason, to average out these small deviances