No of cycles less? - (Nov/17/2005 )

Hi today I did real Time PCR for the 1st time. What i could see is a slight increase in fluorescence during 30th cycle which is the last cycle. so is 30 cycles very less? (dna template is very less as it is the forward primer which acts as template and telomerase will extend if working fine). can we consider that small increase in fluorescence? and also can some one suggest best annealing temp for
TRAP (Kim & Wu) primers(TS and ACX)...

-Molonco-

I am not sure your assay is working. I'll be honest I am not sure what your assay is but you should see exponential rise in curve from 15 to 25 cycles onwards depending on template. Routinely 40 cycles is minimum. Try a free software program such as Primer 3 (you can find in google) for annealing temps and stuff or use Qiagen's online one. Also Qiagen and Stragene do very nice pdf handouts on RT PCR for the beginner.
http://www.stratagene.com/tradeshows/feature.aspx?fpId=1
For qiagen (www.qiagen.com) just browse through the protocols.
Parky

QUOTE (Molonco @ Nov 17 2005, 11:30 AM)

-Parky-

Could you attach a picture of what you are describing? Also, to determine an ideal annealing temperature, consider running gradient PCR to determine this. Good luck.

-LabGirl-

Hey thanx for the replies friends. I got the ampliication this time at 25th cycle itsel.... but annoyingly my heat inactivated -ve control also showed increased ampliication. (In pic green line!). I heat inactivated the protein at 85C for 10'.(telomerase should degrade thereby no template for PCR is formed). What could've gone wrong???
(Red: +ve 50C annealing
yellow:+ve 60C annealing
green: heat inactivated -ve
blue: lysis bufer -ve)

-Molonco-