I have stuck with my rt-PCR problems for months. I have got weak bands or no bands with my pcr products on housekeeping genes like PDH and GAPDH. I have run the RNA on gel. There were 2 distinct bands. I ran a set of samples using master mixes(M) from my stock and my colleague with combination of PDH primers(P) from the superstock and my stock. ie. KM-KP, KM-SP, SM-SP, SM-KP. The bands from KM-KP was so faint. KM-SP was most visible. Brighteness: KM-SP> SM-SP> SM-SP>KM-KP. The concentration of the master mix and the Tm of PCR was followed by the SOP used for years in my lab.
I thought it was the matter of my primers. I replaced my primer with the super stock. I repeated the samples which had weak bands previously including the set of samples(Z) I run the combination of master mix and primers. I got the samples Z with very weak bands and the other with either weak or no bands.
Thanks for attention. I hope you can save my from this despair.
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