Best way to mix qPCR master mix and high ct deviation questions - (Apr/05/2011 )
I am having problems with high Ct deviations of my control gene. I make the master mix by adding:
1. 36 uL of water to each tube
2. 14 uL of a mixed forward and reverse primers
3. 60 uL of EvaGreen Mix
4. 14 uL of a 1:30-1:50 dilution of cDNA
At this point what is the best way to mix the tube and at what temperature? I do my setup at room temperature and was told that doing it on ice can affect the pipette error. The tube never appears homogenized so I flick them a few times and stir with a pipette and do a quick pulse centrifuge to pull down any drops on the side of the tube. I aliquot 20 uL into the appropriate wells (there always seems to be a small bubble on top) and do this for 3-5 replicates (depends on experiment). Yet no matter how careful I am I get deviations that are between 0.3-1 for my 18S control which passes the Ct at cycle ~25ish. Any advice on how to mix the tube so that it is homogenized would be appreciated along with any advice on how I can lower my deviation.
Prepare it in 1.5ml tube and vortex thoroughly, however, 0.3-1.0 deviation near Ct 25 is not a large deviation in my opinion. It might be normal as the PCR is a stochastic process.
vladooo on Fri Apr 8 12:02:14 2011 said:
Prepare it in 1.5ml tube and vortex thoroughly, however, 0.3-1.0 deviation near Ct 25 is not a large deviation in my opinion. It might be normal as the PCR is a stochastic process.
You can vortex with the enzyme? That won't kill it? Everyone here has been saying that I need 0.2 as my replicate deviation. Anyways I will try the vortexing.
Don't be worried about the enzyme, vortex won't kill it, for sure.