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looking for a vector with a Neo cassette, HindIII and BamHI restriction sites (i


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#1 madmed

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Posted 13 May 2009 - 07:47 AM

Hi there,

I've been trying to insert a plasmid into a vector with a Neo cassette using blunt-end ligation and it doesn't seem to work.

My plasmid is digested from a source vector with HindIII and BamHI. Thus I am looking for a vector with a Neo cassette that has these unique cut sites in this order, to try a sticky-end ligation.

If anyone has any ideas, please reply!

Thanks!

madmed

#2 phage434

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Posted 13 May 2009 - 04:39 PM

Find any neo resistant vector and PCR amplify it with 5' overhangs on your primers containing those cut sites. Check that the vector is not already cut with them -- probably it is not. Add 6 bp of junk bases 5' of the cut sites to assure that the RE will cut. PCR, column cleanup, check for a clean band. Digest with your two enzymes + DpnI (digests the parent plasmid used as a template to reduce background). Heat kill if you can, column purify, ligate, go.

#3 madmed

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Posted 13 May 2009 - 08:01 PM

Find any neo resistant vector and PCR amplify it with 5' overhangs on your primers containing those cut sites. Check that the vector is not already cut with them -- probably it is not. Add 6 bp of junk bases 5' of the cut sites to assure that the RE will cut. PCR, column cleanup, check for a clean band. Digest with your two enzymes + DpnI (digests the parent plasmid used as a template to reduce background). Heat kill if you can, column purify, ligate, go.


Thank you, phage 434!

I was looking at other forums like this and I actually found the complementary method to this one, that is, to PCR the source vector (with a focus on my plasmid fragment) and adding to my plasmid the RE sites that are available on the destination vector.

Which of the 2 options is more feasible? PCR-ing the destination vector or the plasmid vector?

Thanks again!

madmed

#4 phage434

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Posted 14 May 2009 - 04:58 AM

Either will work. The second is more conventional, but it is worth noting that the vector is a piece of DNA, not some immutable thing that you cannot change to make your job easier.

#5 madmed

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Posted 14 May 2009 - 02:14 PM

Thank you again!

I'm in the process of designing the primers. Since I'm adding a flanking region of 6 nucleotides, then the new RE site (6 nucleotides), how long should be the portion that will be complementary to the original strand? Usually, one goes for around 20 nucleotides, but when adding the ones above, it comes to 32. All the software I've tried to use to see the Tm tell me the primers are too long.

So how long should that final part of the primers be and still get a good annealing?

Thanks!

madmed

#6 phage434

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Posted 14 May 2009 - 04:17 PM

The Tm that counts for PCR is the part that binds the template. After the first few cycles, the full length of the primer will anneal, but to get started, the 20 bp of the matching sequence has to bind, so that is the Tm that counts. You can sometimes avoid trouble by checking the primer-dimers and hairpin formation with the tools at idtdna.com, or you can just take your chances (which are pretty good). The primer-dimer and hairpins that cause the most trouble are ones where the 3' ends match for 4 bp or so, and where the complementary strand extends beyond the match point. If extension happens, then the primer will no longer bind to the target sequence, but will bind to the incorrect product formed.

#7 madmed

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Posted 15 May 2009 - 09:17 AM

The Tm that counts for PCR is the part that binds the template. After the first few cycles, the full length of the primer will anneal, but to get started, the 20 bp of the matching sequence has to bind, so that is the Tm that counts. You can sometimes avoid trouble by checking the primer-dimers and hairpin formation with the tools at idtdna.com, or you can just take your chances (which are pretty good). The primer-dimer and hairpins that cause the most trouble are ones where the 3' ends match for 4 bp or so, and where the complementary strand extends beyond the match point. If extension happens, then the primer will no longer bind to the target sequence, but will bind to the incorrect product formed.


Thank you! That's what I'm trying to do right now.

I'm thinking about using the Platinum Pfx DNA polymerase from Invitrogen. It's supposed to have high fidelity, works for the size of my fragment (both this one and the next fusion ones). Do you have another one in mind?

Thanks!

madmed

#8 phage434

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Posted 15 May 2009 - 06:08 PM

Our default PCR reaction uses PCR Supermix High Fidelity from Invitrogen. It's a mix of Pfu and Taq. I like the premix because there are fewer pipetting steps, fewer ways to make mistakes, and fewer tubes to contaminate. I usually go directly to an annealing temperature of 55C unless there is a particular reason not to, and it mostly works the first time.




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