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10,000 bp size band showing up in gel of plasmid extraction

gDNA contamination plasmid extraction 10000bp

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#1 tihong10

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Posted 25 February 2013 - 07:01 PM

I am conducting a transformation with a recombinant plasmid. After a round of transformation I thought I had quality transformant, so I performed a plasmid extraction then ran the extraction in a gel. The gel showed a strong intensity band around 10,000bp without any other noticeable bands; but, the recombinant plasmid should only be around 4000. The large size band I guessed was genomic DNA contamination; therefore, I was confused when PCR on the extracted plasmid sample yielded a faint band around the size of the insert. The strange thing is that the transformant gDNA was separately tested through PCR and showed to not contain the insert of the recombinant plasmid.

I am wondering if the large band (10,000bp band) is indeed gDNA contamination i.e. does gDNA usually appear around 10,000bp in 1.5% agarose gel electrophoresis? And also wondering if anyone had ideas on how the PCR on the plasmid extraction yielded a faint insert band when it appeared that only the 10,000bp DNA was present in the plasmid extraction.

Thank You^^

#2 Curtis

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Posted 25 February 2013 - 11:50 PM

maybe your plasmid was a very faint band that you couldn't see in your first gel, but PCR amplified its insert. Do you have any picture of that to upload here? I don't think what you see is genomic DNA. what cells did you transform into? and how do you extract?





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