I prepared a mastermix for technical triplicates for SYBR green assay (reaction volume 10ul)
Out of the triplicates, usually the 3rd replicate will have difference in Ct value i.e. later Ct (range 2-10 Ct difference). As I loaded the 3rd replicate, the volume is
Hello Adam,
Normally 10ul reaction works good. The only set back is we handle very less volume of the components. As you suggested it is mainly due to pipetting error. one thing you can do is prepare the mix in bulk and then aliquote it. it will reduce the handling error.
Good luck.
Yes Ct value will differ if you will use different amount of SYBR mix or cDNA in sample replicate.
When the substrate or enzyme ( ie SYBR mix and cDNA) is limited or completely utilized in any of the replicate, amplification of product is adversely affected dueing PCR and the Ct value differ accordingly however remaining replicate with optimum substrate or enzyme ( ie SYBR mix and cDNA) amplify normally
I always prep for at least 4 extra qPCR wells of master mix, the more wells I am aliquoting to then the more extra I prep, If I need 12 wells I prep for 16, if I need 70 wells I prep for 80, always better to have extra. if you know you are short on one reaction then definitely make a note and exclude it from any calculations.
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