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Very difficult cloning project - how to clone something that is not compatible w


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4 replies to this topic

#1 peachkins

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Posted 13 June 2013 - 07:40 PM

I have a vector, into which I have cloned several genes - finally. All that is left to do is to remove the hsp70 promoter from the vector and replace it with the bactin2 promoter (from another vector i have).

The problem is that the restriction sites (rs) on either side of the hsp70 promoter are SacI, and SalI (and xbaI is right next to SalI), while the rs on either side of bactin2 are ApaI and XhoI. These enzymes are not compatible with each other, and I cannot PCR the destination vector with ApaI and XhoI sites in the primers because there are numerous ApaI and XhoI sites at important places in the vector. I cannot PCR the bactin2 promoter with SacI and SalI site-containing primers because there are SacI and SalI sites within bactin2.

My initial approach has been to simply digest everything with the enzymes for the restriction sites already present in the respective vectors, and then blunt the ends and do a blunt ligation. So far, I have not had success with that strategy (samples getting degraded after T4 usage – even though I inactivate the enzyme, samples degrated after Klenow, even though i inactive it, ligation yielding self-ligated destinated vector… etc), but I am still trying and hopeful.

I’m under pressure from my PI to pursue another cloning scheme along side this, in case it does not work. I thought maybe there would be some way to PCR some compatible cohesive end restriction sites onto the bactin2, but the only SacI-compatible enzyme site that is not found in bactin2 is Bps1286I… and it is not really compatible. It only has two bases of homology.

I would really appreciate ANY advice or suggestions on this!

The vector will be used for microinjection when finished.

#2 phage434

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Posted 13 June 2013 - 08:16 PM

So, you PCR your long vector with different cut sites in the 5' end of the primer. You get to choose whatever ones you want. You PCR your bactin2 promoter with those same enzymes (or compatible ones), ligate, and go. There is nothing magic about your vector which prevents you from using PCR on it in exactly the same way you would use PCR on the insert.

#3 peachkins

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Posted 14 June 2013 - 09:20 AM

Hello, thanks for the response!!!

I guess I should have also mentioned that my destination vector is very large (over 12kb, including hsp70) and that is the other reason I have not tried PCR already.... or is PCR the only way to do this?

#4 phage434

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Posted 14 June 2013 - 10:52 AM

Ten years ago, this may have been an issue. Modern PCR enzymes make a 12 kb fragment relatively easy to amplify. I'd suggest Q5 or Phusion. There are probably other ways of doing this, but I don't think any of them are easier.

#5 GaryJing

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Posted 08 July 2013 - 06:22 AM

You can try cloneEZ or other enzymes like it. Just simply put 15 bp homologus bases at the 5' of your primers. it's very simple and useful.




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