5 replies to this topic

[Curtis]

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?

Edited by Curtis, 23 February 2009 - 07:55 AM.

[merlav]

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.

[Curtis]

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?

[merlav]

I design the primers for QPCR for a length of 125-150bp

[saly]

[/quote]

Hi,
please help me
i made PCR reaction & RT-pCR, the result was as this :

PCR reaction DNA (sample) give band at 150 bp
PCR reaction DNA(negative control) give band at 150 bp
                       But
RT- PCR RNA (sample) give band at 150 bp
RT- PCR RNA (negative control) no band (clear)

** please, what is the Explain of this situation ?
*** this band was primer- dimer or other thing ( as Original band " spesific band" )
thanks for and sorry for elongation
saly4ever@hotmail.com

[littleaxt]

Hi!

Well, if your no-template control in your normal PCR gives you a product you have propably a contamination, whereas your RT-PCR mastermix seemed not to be contaminated.
Or I got something completly wrong, because I wonder a bit about your astonishment.

All the best,

Jan