plasmid construction

Jess via methods%40net.bio.net (by shaakanin from yahoo.com)
Thu May 24 23:20:29 EST 2007


  On 5/8/07, Yvonne Couch wrote:
>
> Hi all,
>
> I am trying to construct a plasmid and I need to take out some loops from
> one plasmid and insert them into pcDNA3. The loops are around 1000bp and
> have BglII and BamH1 at either end so they can be digested out. The main
> problem is the only way I can think of taking the loops out is by
> digesting
> and then running a gel and extracting but this results in tiny amounts of
> DNA that fail to ligate under any conditions.
>
> Any ideas?
>

  Yvonne,
  
I have a couple for you that no one has yet suggested:
   
  If you have a low copy vector, (the one you're taking your loops from) start with 5mL of cells and purify your plasmid from that instead of a regular 1-2 mL starter culture.
   
  Which enzyme system ar eyou usign (NEB? Invtrogen?)- check your buffer compatibility- you may need to do sequential digestion to get good cutting. i.e.- NEB BamH1 has a unique buffer and BglII may not work well in this buffer, even though the catalog says it is the one to use when double digesting.
   
  Digest overnight at 37C instead of just for 2 hours. This will improve your digestion efficiency.
   
  Miniprep kits are notorious for not telling you that heating your gel slice to 65C will give you improved DNA yields. This can help you get a better yield from your gel. (Also if you use the Invitrogen kit, don't use their Elution buffer- its TE and will interfere with subsequent digestion and ligation steps, Qiagen's EB is ok, its just tris-cl. If you are worried about this salt interfering with your digest, then elute with water, but pH it to around 7.5-8.)
   
  When ligating, adjust your DNA concentration (Insert:vector) 90:30 fm ends - this can increase your ligation efficiency.
   
  Try the ligation for 24 hours at 4C, instead of 2 hours at 37, you can also try 18 hours at 16C.
   
  Try also different cells- if you are using DH5a try XL-10. Are your cells competent or supercompetent? To test this, try a positive control next time you transform, just transform vector and plate to see what you get in terms of # of colonies.
   
  And of course, I totally agree with CIAP treating your vector- I would NEVER do a ligation without Cip treating, first ( just remember never to Cip treat the insert, otherwise you'll never get any ligation!)
   
  I hope this is helpful- these are common lab solutions that are useful to us when we have difficulties with DNA ligations. 
   
  Cheers!


    Jess
   
  Grad. Res. Asst.
  University of Texas- Austin
  Institute for Cellular and Molecular Biology
  Department of Medicinal Chemistry
             -Hook 'em Horns!
   



       
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