*Long post*

Dear colleagues,

I am trying to linearise a plasmid fragment of ~6.5kb size by PCR with primers that generate overhangs to then perform a Gibson assembly. It is done with a standard Q5 polymerase 2X mastermix (NEB) as a 25 or 50-uL reaction. Firstly, I ran a normal PCR without any additives like DMSO or messing up with extension times or temperatures. That did not work, but I was able to amplify the inserts of interest with Gibson overhang primers. This was surprising, as these overhang-generating primers, intended for inserts had 2 or 3 secondary binding sites with very low Tm. This was not an issue for the vector-intended primers, but for some reason, this vector linearisation keeps failing.

I then ran a few PCR reactions using recommended annealing temperatures for each primers (Melting temperature minus 3-5'C) with or without 3% DMSO, with or without 2X primer concentrations, and some reactions at at 65'C with extension time of 8 minutes, enough to cover the plasmid twice. This also didn't work.

Finally, I ran a gradient PCR between 72'C-42'C and extension time of 8 minutes without changing primer concentraiton and without DMSO. This also didn't work.

I still have a mix of template, primers and Q5 2X mastermix in the fridge, so will run the gradient PCR one last time, this time my gut feeling tells to give an extension time of 20 minutes and denaturing time of ~1 minute instead of 30 seconds in case if there is some issue with strand denaturation.

However, I just cannot come with an explanation, what goes wrong (positive control works). The template is high-purity, 6.5 kB, 55% GC, at 0.5 ng/uL resuspended in nuclease-free in water with 0.5 ng used in reaction. The Q5 mix is new and verified for quality, the vector-intended primers are ~30 bp long with Tm of the vector-complementary parts approximately Tm=60'C. I have linearised plasmids previously by Q5 PCR no problem, but this particlar vector is stubborn and does not want to linearise by PCR.

Unfortunately, the restriction digst may not be a good solution since the restriction site generates sticky ends that would mess up with the reading frame due to the partof restriction site also acting as a start promoter. It can be fixed by mutagenesis PCR, although it would be the same thing and would probably not work. I designed new overhang primers for the inserts that skip part of sticky end to keep the insert in-frame, but do you think if a Gibson reaction would be set, then 50% of the plasmid would have insert in-frame and 50% would recreate the sticky ends therefore leading to out-of-frame product?

Apoogies for a long post, I am very time and finance-restricted with this project, so ordering 3 plasmids from IDT is not an option.

Any ideas on how to fix this would be greatly appreciated. Thank you for your time,

Kind regards,

Maria