Differences seen in regular and real time machine - (Aug/31/2007 )

Hi,
Running into some issues:

I have 467 bp product reaction in 16SrRNA from bacterial cells. However, while my regular robocycler gives a clean band, use of other realtime machine (to help me quantify)has lead to inconsistent results for same supermix, and dna concentrations.

Reaction details

Initial Denaturation : 96 X 1 min
PCR: 96 59 72 --> for 30 30 30 sec for 40 cycles.

Tm product: 90
Tm primers 75 and 69
Mgcl2 3mM
Primer conc 400 nM each
primer length: 18 and 23
Amplicon length : 467



I have tried doing reactions without SYbr to see if it was inhibiting the reactons, --> didnt find it to be the case..


Suggestions?

Hi Anuyk,
I went to a training seminar on qPCR recently. The optimal conditions give for Sybr assays included Tm 55-60C for the primers and a product of 75-200bp. I'm not an expert but I wonder if your PCR product is too large perhaps.

Best wishes,
Ceri

hmmmm

could you post a pic of the bands? this is often very telling

aside from the length of your amplicon (for good reaction efficiency I was told that you want 50-150bp in qpcr, much like what ceri posted) I also am a little concerned with the differences between your two primers. there is generally much more wiggle-room when designing primers for straight pcr, but qpcr can be pretty unforgiving because everything you're doing is based on the efficiency of the reaction. is it possible to use a primer pair where the two primers are much more similar to each other?