Hi Haaris Ahsan, DNA mutagenesis is pretty much straight forward this days. One can use kit(s) from Agilent or Thermofisher which insure that you have TOP quality reagents or one can do with the same reagents at hand in the lab. Most important point is to have high-fidelity PCR polymerase, so that you introduce only muation you want and not any other. There are many protocols, easiest one is with overlapped primers with mutation you need to introduce over the site. It is usually with primers of 30-36 bp with annealing T about 60-65 C. That's about it. One does PCR with this primers and 10-50 ng of plasmid for 20-25 cycles, than digest original template with DpnI (methylation-sensitive Restriction Enzyme) and transform 2-5 ul of your Dpn1 treated PCR into high competence bacterial cells (no additional inactivation step is needed). Pick some colonies and sequence mutated area, ( so you need to order sequencing primer about - 30-50 bp away from your site). Phusion mutagenesis is a bit more complicated but works better in my hands, you need phosphorylated(non-overlapping ) primers and there is additional ligation reaction before DpnI treatment. In your case Stratagene protocol is first to try. Good luck!

Agree with Dmitry

Hi Haaris

Use Quick change II mutagenesis kit from Agilent. Design your primer using Agilent Primer designing tool for

Quick change mutagenesis.

PL note: Treat ul below for microliter.

Use 25ul reaction with 50ng of template DNA (1ul volume). Add 1 microliter of each primer of 5micromolar conc., 2.5ul Buffer, 0.5 ul pfu ultra polymerase, 0.5 ul dNTPs and 18.5 ul nuclease free water.

Next use these reaction conditions:

95 degree celcius for 2 min.

Do 18 cycles at

95 degree C for 30 sec.

55 degree C for 30 sec.

68 degree C ( use time 1 min/kb plus one minute extra acc. To length of your whole plasmid with insert)

Final extension 68 degree C for 7 min followed by 4 degree C.

Next add 1ul dpnI acc to conc. given in the kit and incubate the reaction for one hr at 37 degree C.

Finally, use 5 ul of this to transform in 50ul bacterial cells e.g. XL-I blue or alpha select cells.

Next morning pick 4-6 random colonies, do colony PCR and send for sequencing to check the mutation.

Most important: Use 68 degree C extension for plasmids greater than 10 kb and 72 degree C for less than 10 kb.

Good luck :)

Hey Haaris,

You can do site directed mutagenesis of this plasmid using Phusion polymerase enzyme and with proper primers containing the mismatch of your interest. Follow the conditions of Phusion enzyme and then after PCR treat it with DpnI enzyme (make sure to put one for DpnI untreated also) and followed by next steps.

Since you know how to design primer from Prof. Ajitkumar class.

Regards