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What I am trying to do is take out a existing promoter region in a plasmid, and replace it with a new one. So first I use the appropriate restriction enzymes to get rid of the existing promoter region. Next, I add in the new promotor fragment which has the appropriate sticky ends, and then through ligation, I want to place the new promotor in the plasmid.

Since the old promotor region and new promotor region will have the same 'sticky ends', how do I prevent the old promotor region from getting placed into the plasmid instead of the new one during ligation?

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  • $\begingroup$ Performing the ligation and sequencing 5 - 10 clones is an option. You only need one clone with the 'new' promoter which will be added to the ligation in excess right? $\endgroup$ – Michael_A Jul 14 '16 at 23:23
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  1. Run it out on a gel after digestion and extract the vector only.

  2. Treat with a phosphatase after digestion. This will prevent religation since you need at least one of your fragments to be phosphorylated for the ligation to work.

  3. (A must regardless) sequence several colonies at the end.

I recommend doing all three.

Edit: option 4: digest with a third enzyme that cuts in your old promoter but not the vector or the new promoter.

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  • $\begingroup$ If your new promoter is short and you are using a pair of oligos with sticky ends that don't need to be cut, you should either phosphorylate the oligos with polynucleotide kinase, or don't dephosphorylate the vector. You need one or the other to be phosphorylated to get the ligation to work. Also, picking many colonies is mandatory. I typically do 24 minipreps at a time because that's what fits in the centrifuge. Do as many as you can. There have been many times where only 1 miniprep was successful. Speaking of which, have a good way to screen, you need nice clear results. $\endgroup$ – user137 Jul 15 '16 at 9:24
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    $\begingroup$ So to confirm, once I get rid of the old promotor region, run it on a gel to isolate the 'opened plasmid with no promotor region'. Once I have this then I preform ligation with the new promotor region.... $\endgroup$ – Adam Radek Martinez Jul 15 '16 at 11:52
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    $\begingroup$ On the plasmid I will have amp. resistant gene as a way of tracking the succesful transformed bacteria. $\endgroup$ – Adam Radek Martinez Jul 15 '16 at 11:54
  • $\begingroup$ @RadekMartinez Yes, you are describing the standard cloning procedure, cut, paste, check for good plasmids. What is your new promoter? How large is it? $\endgroup$ – user137 Jul 20 '16 at 3:12
  • $\begingroup$ I am not sure yet. However it is not like 20 - 30bp, much larger. $\endgroup$ – Adam Radek Martinez Jul 21 '16 at 12:54

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