I am trying to optimize a nested PCR method for qualitative detection of p210 BCR-ABL1 mutation in CML clinical samples. I am following method developed by Tim Hughes and Susan Branford in the book Myeloid Leukemia - methods and protocols. In this book, the PCR method for nested PCR involves the following steps:
a. 1 cycle at 95°C for 10 min.
b. 30 cycles at 95°C for 30 s, 60°C for 30 s, 72°C for 30 min.
c. 1 cycle at 72°C for 7 min.
I have highlighted above that the extension phase seems to be too long. My PCR is not working on known BCR-ABL positive samples. Can extension phase be this long in PCR? Can this be a typo?
The book chapter is attached hereby. The protocol is given on page 84 of the book (Page 94 of the pdf document)
Any help would be appreciated.
Take note of Julie's comments, and also confirm the primer sequences you are using particularly if you are not the person that designed it...maybe from literature
Thanks Julie and Nkechukwu. I did a PCR with 30 seconds time still no results. Will repeat it today. Fingers crossed
Maybe you may need to run gradient PCR to get the best annealing temperature that could facilitate amplification.
We have a real-time PCR kit available. Using that kit, this patient was positive for BCR-ABL and control gene. I am using the same cDNA. The purpose of using conventional PCR is to reduce the cost of the test to the patient.
I am using a Thermofisher Green MasterMix which included all the reagents for PCR and gel electrophoresis. Maybe that's the problem?
Yasar
I just checked the cDNA with GAPDH conventional primers. The cDNA is good.
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