So I began my PCR experiments about 2 weeks ago and everything was going fine and dandy--got my PCR bands, ligation and transformation went well, got my colonies. However, when I did the mini-preps and restriction digest with my enzymes, I see my desired band (at around 462bp) but then I have this faint band at around 2-3kb for both my uncut and cut. And I just have no idea what this could possibly be (genomic dna?). Strangely enough, when I sent the mini-preps for sequencing, the sequence was what I wanted. I have attached a screenshot--first 2 lanes are uncut and cut wild-type and the last 2 lanes are uncut and cut mutant respectively. I've also circled the mysterious bands (2-3kb) in red. Any help will be greatly appreciated!
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#1
furrymittens123
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Posted 02 April 2012 - 07:15 PM
Hello everyone,
So I began my PCR experiments about 2 weeks ago and everything was going fine and dandy--got my PCR bands, ligation and transformation went well, got my colonies. However, when I did the mini-preps and restriction digest with my enzymes, I see my desired band (at around 462bp) but then I have this faint band at around 2-3kb for both my uncut and cut. And I just have no idea what this could possibly be (genomic dna?). Strangely enough, when I sent the mini-preps for sequencing, the sequence was what I wanted. I have attached a screenshot--first 2 lanes are uncut and cut wild-type and the last 2 lanes are uncut and cut mutant respectively. I've also circled the mysterious bands (2-3kb) in red. Any help will be greatly appreciated!
So I began my PCR experiments about 2 weeks ago and everything was going fine and dandy--got my PCR bands, ligation and transformation went well, got my colonies. However, when I did the mini-preps and restriction digest with my enzymes, I see my desired band (at around 462bp) but then I have this faint band at around 2-3kb for both my uncut and cut. And I just have no idea what this could possibly be (genomic dna?). Strangely enough, when I sent the mini-preps for sequencing, the sequence was what I wanted. I have attached a screenshot--first 2 lanes are uncut and cut wild-type and the last 2 lanes are uncut and cut mutant respectively. I've also circled the mysterious bands (2-3kb) in red. Any help will be greatly appreciated!
#2
phage434
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Posted 03 April 2012 - 05:09 AM
I'd vote for supercoiled uncut vector. Sometimes it is very hard to cut completely, and this may be a case. I would go forward with your experiments and not worry about it.
