1 reply to this topic

[sk2775]

Hi,

I am getting Ct around 30-32, and it was suggested to me that if I increase my primer concentration a bit, it may lower the Ct.

Currently, my master mix solution is made such that I use
10 uL SYBR Green
0.25 uL Forward Primer (@5uM)
0.25 uL Reverse Primer (@5uM)
4.5 H20.

What is the best solution to try to optmize to lower Ct a bit.  Is it by may be increasing my primer concentration to say around 10 uM.

Thanks.

[zowawih]

You did not mention what the DNA concentration of your tested samples. If you are using samples with high concentration you should get Ct value lower than you've got. If you are getting curves at low Ct and other at 30-32 that can be primer dimmer. At the same time, YES the low primer concentration result in low Ct value. It’s all about trying and optimizing.  
I would till you give it a go. Try to increasing a bit and decreasing.