Real-time PCR problems
Posted 04 March 2005 - 06:44 AM
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
i use an iCycler with Abgene's SYBR Green Master Mix with Fluorescein in a 25ul volume, 2.5ul template, 70nM final primer concentration
Posted 04 March 2005 - 07:37 AM
Posted 04 March 2005 - 11:59 AM
again any help is very much appreciated!!
[ to test my [pipetting i'm goping to run 10 wells of the same sample next week ]
Posted 04 March 2005 - 07:17 PM
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.