Unexpected bands for negative controls of my PCR on the gel

forum123 · #11 03-14-2011, 09:14 AM

hey all, I also have the exact same problem... can I know the best way to prevent contamination?

lisalin · #12 03-16-2011, 01:43 PM

Quote:

Originally Posted by forum123

hey all, I also have the exact same problem... can I know the best way to prevent contamination?

Just throw away the old reagents, and purchase new ones. Don't waste ur time to find out which reagent was contaminated. Use alcohol to wipe out the surface of laminar flow hood and then UV sterile the hood together with other PCR supplies (but not reagents!!) such as pipet, tips, tubes, even gloves before every PCR trial. At least, these work for me

LunaLu · #13 05-19-2011, 05:14 AM

Quote:

Originally Posted by butters

is the unwanted band as intense as ur positive control? or very faint but slowly increasing when more pcr is perform? where u load ur DNA?

Hi...the positve control of my PCR showed non specific bands slowly increasing when more PCR i perform.....do you have any idea why this is happening

butters · #14 05-20-2011, 05:52 AM

Oh, my its been ages since I come here.

Back to point. Slowly increasing in PCR band intensity mean introduction of more template from the previous reaction aka carry over contamination.

You did mentioned positive control right? What about your negative control? Well, I would "bang" on the part you use the same pipette for loading your positive control template right? A plasmid maybe?

LunaLu · #15 05-22-2011, 02:35 AM

Quote:

Originally Posted by butters

Oh, my its been ages since I come here.

Back to point. Slowly increasing in PCR band intensity mean introduction of more template from the previous reaction aka carry over contamination.

You did mentioned positive control right? What about your negative control? Well, I would "bang" on the part you use the same pipette for loading your positive control template right? A plasmid maybe?

Thanks for answering me…

The negative control of my PCR doesn't show nonspecific bands on the agarosa gel BUT always appears a band with same size as the target band (164pb)

…
And yes, I use the same pipette for loading the DNA template

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Unexpected bands for negative controls of my PCR on the gel

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