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smear in negative control brighter than in samples


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#1 sara27

sara27

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Posted 18 November 2010 - 05:04 PM

Hey all,

I've been fumbling around cloning for some time now but have reached a result I'm not sure what to do with. I am trying to do a PCR from a yeast genomic extract and at first set up all my samples and a negative control. After running on the gel I saw a band that was too small to be my product in my samples and a very light smear extending from the well to the band and a very light smear in the negative control (PCR master mix with water) that continued past the band in the other samples. I repeated the PCR since my template is kind of tricky and has a GC rich region, adding DMSO to it. Now I have no bands in the PCR reactions with samples, just a light smear akin to what I saw in the negative control the first time, and a smear about twice as intense in my negative control this time. I already changed the water between my first and second attempts and use filtered pipette tips to set up the reaction, and my lab mates are running PCRs with the same reagents with no problems so I can't figure out what this smear is or how to get rid of it. Any ideas would be highly appreciated!

Thanks in advance :)




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