I am trying to assemble a plasmid for transfection by Gibson assembly. Because there are no unique restriction enzyme sites in the region I am trying to clone into, I am effectively trying to linearize my plasmid by PCR with two primers designed back to back (they were selected by the NEB gibson assembly program). I have tried conducting the PCR using both Herculase II Fusion polymerase (from Agilent) as well as the NEB Phusion high fidelity polymerase. In both instances, I get random bands that are shorter than the total size of my plasmid, suggesting that perhaps the primers are binding to another location in my plasmid, or there is a point where the enzyme may stop polymerizing.
Does anyone have any tips on how to check for these? or any suggestions for how to minimize unwanted bands?
The vector I am using was generated by TA cloning a previous PCR product into pGEMTez vector.
I don't know how much effort it took to generate the plasmid with the first insert, but I would consider to start again with an empty vector and two PCR products. Always try to generate constructs with at least one unique restriction site flanking each end of your PCR products.
If the primers designed are too close, you could have problems amplifying circular plasmids.
Make sure the primers are right, even if they are selected by a software. Some software may design plasmids with too many or key nucleotides "mutations", making the plasmid not bind.
You may design short primers by yourself, with restriction enzymes, force blunt ligation, transform it and keep it in a plasmid/cell bank in -80. So, whenever you need the vector again it has restriction enzymes to linearize it. You can also use this approach for to have the linear plasmid and then use the primers the software design.
An PCR approach is Touchdown PCR, where you do 5 to 15 cycles decreasing 1or 2ºC on the Annealing Temperature, every cycle. Then you run a normal PCR on the lowest Annealing Temperature reached. This approach tends to amplify the right fragment instead wrong ones (As long everything is ok with your primers).
I had these problem last time, amplify the plasmid and did not obtain the size expected. We changed the Taq (specifities are differents), do touchdown PCR to check the better condition, dilute the template...or simply, change the primers: I design myself and check if there is no hairpin formation or dimerisation....
Actually I have some primers that work in most plasmids if you want to try them. I know I'm a little late in replying, but let me know if still interested.
Hi Kimberly
How did you finally manage?
I have the same issue now and it's frustrating.
Thank you
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