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Double band in PCR screen of homologous recombination in yeast

Double band in PCR screen of homologous recombination in yeast - Yeast Forum

Double band in PCR screen of homologous recombination in yeast - Discuss research on yeast the model organism. Post questions and topics on the molecular biology and genetics of Saccharomyces cerevisiae and other yeast species.


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Old 01-28-2011, 05:18 PM
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Default Double band in PCR screen of homologous recombination in yeast



Hi there,

I'm doing homologous recombination in yeast and have got a rather strange result, and I was hoping someone could shed some light on it. To screen my yeast for positive recombination, I use PCR primers flanking the recombination site and should get a 2kb band on my gel if there is no recombination, and a 1kb band if recombination has occurred. The 1kb band encodes phleomycin resistance gene.
Weirdly though, I am getting double bands in a lot of my colony PCR screens. I get both the 2kb and the 1kb band. I even took one of the colonies that did this and streaked it out, screened those colonies, and got the same problem again.
Has anyone ever encountered this, or does anyone know what's going on?

Thanks,
Cat.
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Old 07-17-2011, 08:20 PM
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Default Re: Double band in PCR screen of homologous recombination in yeast

Hi Cat,

I was wondering whether you have already solved the mystery? By the way: do you also have colonies that only show the 1 kb band? How does your wildtype control look like?
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Old 09-08-2011, 01:54 PM
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Default Re: Double band in PCR screen of homologous recombination in yeast

Nope. Never got to the bottom of this one. No colonies showed the 1kb band. The wild-type control had just the 2kb band.
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band , double , homologous , pcr , recombination , screen , yeast


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