High Ct values - (Dec/21/2009 )
Hi everyone,
I am working on mouse ES cells. Currently using 50 million cells as per the protocol. Even then I am getting the high Ct values (around 35s) in a 383 well QPCR machine. I have not quantified my DNA samples after pull down with respective antibodies. I am using just 10 ul of the total DNA isolated. Why am I getting High Ct values. Can anyone please tell where am I getting deviated.
Depends on many parameters, you need to give us more details of what you do. I used to get terrible Cts also, but I modified many things.
my method was SYBR Green, later I used more buffer. or I used more primer concentration.
if your DNA sample conc. is ok then the pro is not from your samples.
have played with your machine's GAIN? because your result can be different from machine to machine. usually plate based machine's give higher Ct, go for a Corbette RotorGene machine. it works much better and more precise.
sameermahmood on Dec 21 2009, 09:42 AM said:
I am working on mouse ES cells. Currently using 50 million cells as per the protocol. Even then I am getting the high Ct values (around 35s) in a 383 well QPCR machine. I have not quantified my DNA samples after pull down with respective antibodies. I am using just 10 ul of the total DNA isolated. Why am I getting High Ct values. Can anyone please tell where am I getting deviated.