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PCR troubleshooting - no product

PCR troubleshooting - no product - PCR - Polymerase Chain Reaction Forum

PCR troubleshooting - no product - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 06-30-2010, 11:34 PM
Pipette Filler
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Default PCR troubleshooting - no product



Hi everyone,
I have Drosophila cDNA clones that I prepped using the Qiagen miniprep kit. From this cDNA I am attempting to make dsRNA for RNAi experiments in S2 cells. I have primers targeted against a 600bp stretch of the cDNA sequence, with the T7 promoter added so that I can use the PCR product to generate dsRNA using a T7 kit. Most of my cDNA targets worked beautifully, a single PCR product was generated which I was able to use for in vitro transcription, but one of them just doesn't give me any product by PCR and I can't figure out why. The primers are the same as those used by someone else in the literature so I know they have been successfully used in the past, and I have checked using software to make sure I should only get one product and that there are no primer dimers etc. I've tried fiddling with the annealing temp, both increasing and decreasing, and the extension time and number of cycles, to no avail. The last time I did the PCR I did get two very weak products, but at completely the wrong size, much larger at around 6kb and 3kb. Does anyone have any suggestions for where I might be going wrong or how I could troubleshoot this?
Thanks!
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