Hello every body. 

Using (SensiFast Sybr No-ROX one step kit) from Bioline, I did qPCR experiment used one -same- sample with 3 different concentrations of RNA and two different reference genes. Concentrations used 300 ng in 20ul reaction, 400ng and 500 ng.

The reaction in wells A, B and C in the attached images included the master mix according to the procedure and the steps as mentioned in the kit. 

D and E include the same sample of concentrations (300ng and 500 ng) the difference is that the RNA was treated with 2ul DNA-wipe for 2 minutes before adding the Kit master mix.

My lab mate used the same samples I used in this experiment, but she used the 2 steps kit (prepared cDNA and then qPCR) and she got nice melting curve! 

Can you please discuss with me this issue? all suggestion will be appreciated.

Thank you in advance

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