Real-time PCR problems - (Mar/04/2005 )

hi all,

i have recently started to use an iCycler for setting up my real-time pcr systems and have encountered problems when trying to run a serial dilution of a template to evaluate my reaction efficiency.

what happens is, i'll make up for example 5 dilutions (1/10, 1/100, 1/1000 etc) of my template but the Ct values which i obtain at the end of the reaction never seem to match (i.e. the 1/1000 sample has the same Ct value as the neat template)... usually 2 or 3 of the samples will have differences in their Ct values which you would expect to see, but it never goes totally to plan.

am i suffering from contamination? poor pipetting? bad primers? (the melt curves are fine)

if anyone has a protocol i can follow i'd be grateful, details of the reagents i use to follow

regards

james brown


i use an iCycler with Abgene's SYBR Green Master Mix with Fluorescein in a 25ul volume, 2.5ul template, 70nM final primer concentration

Just had a similar experience, the standard curve looked great till I got to the final two dilutions which gave me the same Ct value. Afraid I don't have any advice on how to fix it except to start all over and hope for the best.

does anyone know if using a ten-fold serial dilution is the best idea??? the Ct values are all quite low so shall i try to use more template or maybe a series of 3-fold dilutions???


again any help is very much appreciated!!

[ to test my [pipetting i'm goping to run 10 wells of the same sample next week ]

We only do 3 fold serial dilution when performing the standard curve. We usually do this do measure our primer efficiency. I think you are simply having a problem with scale. Sounds like the dilutions are the far end (high cT) are just to small to register a difference in cT. We use five 3 fold dilutions values. They are shown below:

150 ng
50 ng
16.67 ng
5.55 ng
1.85 ng

Of course if you are looking for copy number, these values may not be accurate for your application. However, if you are just measuring primer efficiency this numbers will work just fine.

Greg