Real-Time PCR primer vs conventional PCR primer - (Feb/23/2009 )
why can't we use the same conventional pcr primers in real-time pcr? and why the target sequence in real-time pcr should be less than 200 bp?
-Curtis-
Short amplicons yield the most consistent results. Remember that there is no extension so, the amplification will be incomplete if the amplicon is too big. You can use the same primers for QPCR and PCR if they fulfil the requirements of Tm, amplicon size, %GC and no G's at 3', etc. etc. for QPCR. I had used the same primer set for both, but I dilute the primers working solution for PCR in TE and the ones for QPCR in water because the QPCR reaction is more sentive to salts than the conventional.
-merlav-
merlav on Feb 23 2009, 12:09 PM said:
Short amplicons yield the most consistent results. Remember that there is no extension so, the amplification will be incomplete if the amplicon is too big. You can use the same primers for QPCR and PCR if they fulfil the requirements of Tm, amplicon size, %GC and no G's at 3', etc. etc. for QPCR. I had used the same primer set for both, but I dilute the primers working solution for PCR in TE and the ones for QPCR in water because the QPCR reaction is more sentive to salts than the conventional.
thank you very much, how long is your target sequence?
-Curtis-
I design the primers for QPCR for a length of 125-150bp
-merlav-