Ronit’s Notebook: qPCR with Desiccation + Elevated Temp. Samples (DNMT1)

Before the long weekend, I ran a qPCR assay with my desiccated + elevated temp. samples. There were 40 samples in total and I ran one plate with DNMT1 (DNA methyltransferase) as my gene target.

Plate Layout:

The plate is laid out with duplicates in adjacent wells and the order of samples is as follows:

D01, D02, D03, D04, D05, D06, D07, D08, D09, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, T01, T02, T03, T04, T05, T06, T07, T08, T09, T10, T11, T12, T13, T14, T15, T16, T17, T18, T19, T20

There are 2 positive controls with gDNA in wells G9 and G10. There is a no-template control (negative control) with no template in wells G11 and G12.

To run the qPCR assay, I created a mastermix with 20 µL of forward primer, 20 µL of reverse primer, 400 µL of 2x qPCR master mix, and 320 µL of DEPC-treated water. 19 µL of the mastermix was put into each well and a subsequent 1 µL of cDNA was put in for each sample (water for the negative controls and gDNA for the positive controls). Note that all sample cDNA used was a 1:5 dilution.