Design primers using NEBaseChanger. Also check if the PCR elongation time is optimal for the plasmid (12kb vector ) you are using. 

It might be much simpler and quicker to get your work done by re-designing your primers, just using the DNA polymerase and DpnI from that kit and following the modified protocol described http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91. This modified protocol explains the problems of the primer design used by some site-directed mutagenesis kits.

The attached file is my lab protocol summarised based on that publication and it is much easier to follow.

Thank you very much sir for sharing this wonderful information. I will update you the results of using this protocol.

We now use this routinely though only for more complicated procedures which involve changing more than a handful of bases e.g adding or deleting exons to yield other transcript variants. I would recommend using KOD Hot Start rather than the Q5 provided in the kit (follow KOD protocol for amplification). Aim to have 60oC Tm for the binding section of both oligos (anneal of 60oC during PCR) and have the 90 bp to be added split so each oligo has a 45bp overhang ready for the KLD reaction following PCR. The homologous regions of the primers should be at least 22bp given the length of your overhangs. Reduce the number of cycles to 17 and use a 5-6 minute extension time. Using these guidelines works well for us. Good luck! 

Since you want to add a tag, I might be simpler to reamplify the insert, while adding the tag with duely designed reverse primer. If you insist on using site directed mutagenesis, you could try designing/checking the primers in "PrimerX". This on-line tool would help circumvent design issues

From my own experience, NEB Q5 Site Directed Mutagenesis Kit works like a charm - but mainly when you have a relatively small vector (~ 5 kb). 

I had a similar problem when I tried to introduce mutation in my ~600 pb insert in the 9.2 kb vector. I switched the strategy for initial cloning of my site directed mutagenesis target into a smaller vector (I use pUC18; ~2.6 kb) and after introducing the mutation I subclone the derivative insert into my final, big vector.

Hope this will work.

Hi, try the Fastpfu or Fastpfu FLY from Transgen Biotech. They always work... I just achieved something similar with 36%GC primer binding sites and a lot of floating stuff ! 

Hi.

I've used the Q5 kit to generate many different mutants, but in my experience it works well only with point mutations and small insertions, using primers exclusively designed with their web tool NEBchanger. I've noticed that the polimerase messes up when the primers have very high chance of forming self- or hetero-dimers, so in your case I would not recommend this kit. I think the best strategy is to redesign your Reverse primer in order to have a much more similar Tm to the Forward and just avoid the annealing during the PCR, going straight to the elongation. Another possibility is that you place the insertion in subsequent steps. I believe you might have higher chance of success if you do it in steps...

Dear Suyash Patil.

You have got already many valuable suggestions, especially a protocol from Huanting Liu. We also use it a lot. However, there are several additional aspects to consider.

Insert. You have mentioned your insert is 90 bp long and it's repetitive. Why not to consider ordering synthetically produced double stranded piece. In many cases it's a bit more expensive than just a pair of primers but you solve a lot of problems. Many companies provide these under different names: IDT- gBlocks, GeneArt - DNA strings, eurofins has something similar.

DNA Polymerase. In principle you don't need any kit for mutagenesis today. The only requirement is proof-reading dna polymerase, better with hot-start ability. You would like to have both to avoid mistakes during reaction and have it active only when you start the reaction in PCR machine and not when you are preparing it on the bench. However, any mutagenesis protocol assumes that you perform whole plasmid PCR, which might be difficult for some enzymes. In your case you might not be able to amplify the whole plasmid correctly and that's why you don't have any product. In our hands the following enzymes has been used: KAPA HiFi (Kapa Biosystems), SuperFi (Thermoscientific), Q5 (NEB), KOD and Pfu Ultra. In addition, I would recommend to use their ready mixes. They are a bit more expensive, but it reduces the number of manipulations and as a consequence the number of parameters, where you can make a mistake. Also, we scale down all the reaction volumes to 10-25 ul, and then you actually make it even cheaper.

Other methods. As already suggested, you may use two step approach to add your tag and then reclone it back, by e.g. using restriction enzymes. This way you avoid the amplification of the plasmid in vitro, in case this is a problem.

Just a note. I assume you use DpnI to digest the parental plasmid, that is used as a template in your reaction. I would recommend to use FD-DpnI from Thermoscientific, as their enzyme appears to be much more active in different buffers. We just add it directly to any polymerase reaction and it works much better than any other DpnI from other suppliers. 

I swear,  get AP221 or AP231, they're hotstarts,  from transgen biotech. If it doesn't work,  I'll do it for you. 

It's unlike anything you've ever used! Outperforms Q5,  Phusion and KOD by far

Dear All, Thank you very much for your valuable suggestions !!