I am trying to amplify a vector by pcr. I linearised the vector with EcoRI and BamHI to ensure no re-ligation. I then purified the linearised vector and used that in my pcr reaction. I am expecting a product around 6.1kb, but I repeatedly get a product around 0.5kb. My primers are good, they are at unique sites and have Tm's of 68 degrees (I am using a 2-step reaction). I use 3% DMSO in the reaction, 10 ng of template vector and 0.5uM F.C for my primers. My primers have 25 bp overhangs homologous to the insert I plan to assemble into the vector. Those overhangs are not homologous to any region in my vector. So far I have tried varying the annealing temperature in both 2-step and 3-step pcr reactions by the touchdown pcr method and just having a single annealing temperature and I have tried varying DMSO %. Can anyone give me advice on what might be happening, and how I might be able to amplify the full 6.1kb. Thanks.
Hi Jacob,
Just a small doubt, after you do the double digest to produce non ligating ends for your linearized vector, are you sure that the fragment that was excised is free of you primer binding sites. 6.1kb is quite a bit to amplify, and in case your primers are binding in a region that still is in the linearized vector try using a high fidelity polymerase.....like Phusion polymerases or Pfu (no proofreading thus higher probability of mutations while amplifying such a long fragment).
Also if your vector is AT rich you may need to revise the thermal cycle.
Good Luck
The primer binding sites are definitely not in the excised fragment and are approx 100bp from the ends of the linearised vector. I have tried Taq and Phusion polymerases and the vector is 39% AT, so I would think that standard thermal cycles should work. I tried another round of PCR using an annealing temperature of 60 degrees to see what I would get and still only have the same 1 product at 500bp.
I have sent the PCR product for sequencing now, using the same primers as for the PCR, so I will see if that works and if it tells me anything.
Hi Jacob,
In my experience, the times I obtained a ghost band of much lower molecular weight that desired, the target band was always missing. In my case however, we figured out the cause to be some contaminating DNA and therefore repeated the entire protocol with all equipment washed in SDS and distilled water. It does sound trivial yet, did cause quite a hassle.
- Badges
- Science method