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I get band double the size what does it mean


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#1 jbourne

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Posted 17 July 2013 - 10:12 AM

hello guys I am new to this forum

I have a problem, I transformed my gene into pichia using pPICZalpha vector , i found that the yeast is growing on zeocin plates n i confirmed that my vector has been inserted into it
to confirm the transformation I ran PCR usin 3' n 5' primers along with a control.

I expect to get a band of same size as that of the control but somehow i see a band which is double the size of the control band

my question is what that implies

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#2 bob1

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Posted 17 July 2013 - 01:32 PM

It could mean that you have a double copy of the gene in the plasmid, or it could mean that your PCR is non-specific (i.e. there are other binding site in the vector), or it could mean that the sequence you thought was in there is actually something else.

Were the primers specific for the insert or were they off the vector?

#3 jbourne

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Posted 18 July 2013 - 06:36 AM

It could mean that you have a double copy of the gene in the plasmid, or it could mean that your PCR is non-specific (i.e. there are other binding site in the vector), or it could mean that the sequence you thought was in there is actually something else.

Were the primers specific for the insert or were they off the vector?


the primers were specific for the vector

#4 bob1

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Posted 18 July 2013 - 12:57 PM

Ok, have you tried getting the product sequenced then?




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