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So I am in a bit of a time constraint. Essentially, I inserted a DNA fragment via molecular cloning which contains a unique RE site. I need to confirm whether my colony has or does not have the fragment. I need to do this in one day so would this work as a confirmation strategy:

  1. Pick colony and perform colony PCR with primers around RE site.
  2. Cleanup the PCR product and digest using the enzyme which was inserted.
  3. Run on a gel. If a cut is noticed, this confirms that the colony had the inserted fragment (containing the RE site).

Would this strategy work? Are there any notable issues people can for see in this strategy?

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One issue is if your fragment amplified is too small you may not be able to see the positive result of two bands because they run off the gel. Also the RE may not cut a small fragment efficiently.

I would recommend extracting and purifying plasmid, then do a double digest using the unique fragment cutting RE and a RE that only cuts the backbone. You should either see linearized plasmid DNA or two bands that are probably both >1 kb and easily resolvable.

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  • $\begingroup$ I would question the cutting efficiency since often PCR amplified products have restriction ends and are easily cloned into vectors. $\endgroup$ – Adam Radek Martinez Oct 16 '18 at 0:37
  • $\begingroup$ Maybe it depends on how far away your primers are but according to this NEB article you need to some extra DNA sequences on both sides of the cut site for efficient cleavage neb.com/tools-and-resources/usage-guidelines/… Since you didn't specify I assumed the worst case where your cut site is at the very end of the amplified product. $\endgroup$ – Cell Oct 16 '18 at 2:02
  • $\begingroup$ Yes, usually its 4 to 5 bp..... my "double check" primers would amplify 500bp from each side of the RE site. It should cleave well. In addition 1,000bp vs 500bp would be very noticable $\endgroup$ – Adam Radek Martinez Oct 16 '18 at 14:29

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