looking for a vector with a Neo cassette, HindIII and BamHI restriction sites (i
Posted 13 May 2009 - 07:47 AM
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!
Posted 13 May 2009 - 04:39 PM
Posted 13 May 2009 - 08:01 PM
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?
Posted 14 May 2009 - 04:58 AM
Posted 14 May 2009 - 02:14 PM
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?
Posted 14 May 2009 - 04:17 PM
Posted 15 May 2009 - 09:17 AM
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?
Posted 15 May 2009 - 06:08 PM