12 kb is really quite long and I would guess might be the cause of your difficulties. Can you try using a smaller plasmid. If not, are you allowing sufficient polymerization time for such long product.

in such case, you should not use the method you mentioned.

I induced two mutations and one 3HA tag in a 11kb plasmid step by step, each time to induce one mutation and using some close restricted enzymes. if I understand correctly, your mutations are very close, so choose two restricted enzyme, and using overlap PCR to induce your insertions, and then ligated it with vector. it will work easily.

if you still want to use inverse PCR to create mutations for such big plasmid, try some commercial kits

http://www.creative-biogene.com/Easy-Change-174-XL-Site-Directed-Mutagenesis-Kit-SMK-Y001-1232919-98.html

Hi Sehar you might try our paper

https://www.researchgate.net/publication/223980074_Improvement_of_PCR_reaction_conditions_for_site-directed_mutagenesis_of_big_plasmids

best regards

you need to used HPLC purified Primers

Article Improvement of PCR reaction conditions for site-directed mut...

@ Michael J. Benedik. Its my lentivirus construct. I am afaraid using a smaller size palsmid is not an option . and yes extention time is min/Kb according to protocol. so i am using 68C-12min

@Shui Sailan, I am creating 2 mutations (6bp each in CDR of antibody light and heavy chain) and restriction sites are not that close. unique cutter is few hundred base pair apart. and i need lot of PCR product to make a bacterial library. so restriction digestion is not a suitable alternate for me.

https://www.researchgate.net/publication/223980074_Improvement_of_PCR_reaction_conditions_for_site-directed_mutagenesis_of_big_plasmids

Article Improvement of PCR reaction conditions for site-directed mut...

thank you all for your valueable suggestions. My PCR is still not working. i have tried different Tm but no luck!