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How to Map a Plasmid by Agarose Gel Bands

BioTechniques Staff

Does my insert have an internal EcoR1 or Sal1 site?

The BioTechniques Molecular Biology Forum brings bench scientists together in an online community to ask for and share advice regarding laboratory problems. This week’s highlighted question comes from the RNA Methods forum.

Q: I have a miniprep pGEX-6P-3 plasmid with a 0.75 kb insert. The insert was cloned into the vector with EcoR1 and Sal1 restriction endonucleases. When I run miniprep DNA from this clone and cut it with either only EcoR1 or Sal1, expecting one band at 5650 bp (pGEX is 4900 bp long), I come out with a band at 1500 bp as well (double the length of the insert). When I do a double digest, I get a band at 4900 bp as well as a band at 750 bp that is brighter than expected. Does this mean my insert has an internal EcoR1 or Sal1 site? Or both?

A: It seems to me that your first instinct was right on...

A: I have some "plasmid troubles" as well. Although mine is not exactly the same, I didn't want to start new topic and hope I can get an answer here...

A: Do you actually have a 600 bp band, or you have a problem telling you that a 600 bp band is generated by PCR?

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