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cant get any PCR products and smears on all lanes

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#1 mitooo



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Posted 23 April 2010 - 06:49 AM

Hi all, I'm currently working on bisulphite sequencing and i'm up to the part where i clone my gene fragment into pGEm vector. When i run my colony PCR, i always see smears on my lanes including the negative control. Sample from blue colony gives me a band of the non-insert size, however nothing was seen in the samples contain template....My supervisor suspected some levels of contamination and we tried to get fresh reagents, the results still look the same....We switched to using premix, primers from different lab, different PCR machine but still the problem couldnt be solved. We even tried to use pUC 19 plasmid and run PCR, we could only see smears...We use M13 primer and you would expect to see a product with puC 19 right? I only have about 5 more months to run this exp and i still couldnt get it going, thus any suggestions would be very helpful. Thank you very much

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