How many cycles in PCR would you recommend for ChIP - (Aug/16/2007 )
Hello,
I have just registered to this forum and became addicted quite soon.
I have started ChIP protocol for the first time in my lab and have a lot of questions.
But first, how many cycles is best for the PCR to get a good signal from the IP DNA?
I have been doing 27 but IP is still not strong.
thanks for the upcoming answers 
-ehallacli-
QUOTE (ehallacli @ Aug 16 2007, 06:17 PM)
Hello,
I have just registered to this forum and became addicted quite soon.
I have started ChIP protocol for the first time in my lab and have a lot of questions.
But first, how many cycles is best for the PCR to get a good signal from the IP DNA?
I have been doing 27 but IP is still not strong.
thanks for the upcoming answers
I have just registered to this forum and became addicted quite soon.
I have started ChIP protocol for the first time in my lab and have a lot of questions.
But first, how many cycles is best for the PCR to get a good signal from the IP DNA?
I have been doing 27 but IP is still not strong.
thanks for the upcoming answers
Technically, your CT values for your target region should be reproducibly lower than your negative controls. For some IPs I get CT values around 25, for others they are as high as 33-34; but my negative control CT values are general in the 35+ range.
-jonathanjacobs-
QUOTE (jonathanjacobs @ Aug 17 2007, 02:28 PM)
QUOTE (ehallacli @ Aug 16 2007, 06:17 PM)
Hello,
I have just registered to this forum and became addicted quite soon.
I have started ChIP protocol for the first time in my lab and have a lot of questions.
But first, how many cycles is best for the PCR to get a good signal from the IP DNA?
I have been doing 27 but IP is still not strong.
thanks for the upcoming answers
I have just registered to this forum and became addicted quite soon.
I have started ChIP protocol for the first time in my lab and have a lot of questions.
But first, how many cycles is best for the PCR to get a good signal from the IP DNA?
I have been doing 27 but IP is still not strong.
thanks for the upcoming answers
Technically, your CT values for your target region should be reproducibly lower than your negative controls. For some IPs I get CT values around 25, for others they are as high as 33-34; but my negative control CT values are general in the 35+ range.
Thank you very much. I guess you are doing real time PCR. Unfortunately I do not have that chance. Hopefully in the future.
-ehallacli-
QUOTE (ehallacli @ Aug 17 2007, 10:37 AM)
QUOTE (jonathanjacobs @ Aug 17 2007, 02:28 PM)
QUOTE (ehallacli @ Aug 16 2007, 06:17 PM)
Hello,
I have just registered to this forum and became addicted quite soon.
I have started ChIP protocol for the first time in my lab and have a lot of questions.
But first, how many cycles is best for the PCR to get a good signal from the IP DNA?
I have been doing 27 but IP is still not strong.
thanks for the upcoming answers
I have just registered to this forum and became addicted quite soon.
I have started ChIP protocol for the first time in my lab and have a lot of questions.
But first, how many cycles is best for the PCR to get a good signal from the IP DNA?
I have been doing 27 but IP is still not strong.
thanks for the upcoming answers
Technically, your CT values for your target region should be reproducibly lower than your negative controls. For some IPs I get CT values around 25, for others they are as high as 33-34; but my negative control CT values are general in the 35+ range.
Thank you very much. I guess you are doing real time PCR. Unfortunately I do not have that chance. Hopefully in the future.
Your best bet is to always run a few PCRs with differing dilutions of DNA to make sure you are in the linear range with your primers (assuming you are doing densitomitry).
-KPDE-