RT PCR for larger amplicon - (Nov/02/2006 )

Hi All,
I am interested in finding the dilution endpoint using nested primers amplifying a ~1.2Kb fragment. What I am planning is to use the first round regular PCR product as template for the second round RT-PCR(real-time PCR)…I want to do the second round using real time with sybr green. I don’t need a probe because I don’t need the specificity, I’m just looking for the lowest dilution of template that will give me a positive PCR reaction.

I have tried this with the AB SybrGreen Master Mix and adjusted thermalcyling conditions…with crappy results.

My question…can I use my usual nPCR reaction components (Fusion polymerase etc, not syber green master mix)) and just add sybr green to the reaction?

Thanks for any input!

mars

Dear girlscientist,

Just to remind you that real-time PCR should not be confused with reverse transcriptase PCR (RT-PCR)

Yes you can use with you normal PCR component.
I used a 1:100,000 final dilution (in dimethyl sulfoxide) of SYBR Green I ,
250 nM concentrations of ROX passive reference dye (in dimethyl
sulfoxide). The rest remain the same.

Dear girlscientist,

I beleive that the 1.2kb amplicon is slidely too large to be able to amplify exponentially.
You might have to increase your dNTP amount.