Dear Arun, I find stratagene method using Pfu turbo enzyme much more successful then using phusion. If you go to their website they even have a tool to design the 'perfect' primer!
Happy cloning! On 24/02/2012 10:54, "Tim Gruene" <[email protected]> wrote: -----BEGIN PGP SIGNED MESSAGE----- Hash: SHA1 Dear Arun, it's been a while for my last PCR, so take my comment with caution: 33bases seems very long for a primer especially if it is only for a point mutation. Could you trim it down to 20-25 bases? I would cut at the downstream side. Tim On 02/24/2012 10:05 AM, Arun Kumar wrote: > can any one help me in suggesting that what mistake i have did in my > mutagenic pcr . actually my problem is my primer annealing > temperature is 81degree. im using phusion pol enzyme. i have made > many trial, i.e., made annealing at 68 and then followed 2 step pcr > method and then added 1micro lit of dmso to 50micro lit of pcr mix > etc.. but till now i couldn't get my desire point mutation. my primer > length is about 33 and the mutation id at the centre of the primer. > can anyone help me what i can improve to get result or what mistake i > had did.. thank you all the members in advance, cheers, Arun > - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -----BEGIN PGP SIGNATURE----- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPR2xKUxlJ7aRr7hoRAuyaAKDcK3tiB1wPtJfwZp3MyTLiP4WsYgCdHZLG YHIJVHXQdxXKlrI+65NeQhc= =YJ5D -----END PGP SIGNATURE----- NOTICE AND DISCLAIMER This e-mail (including any attachments) is intended for the above-named person(s). If you are not the intended recipient, notify the sender immediately, delete this email from your system and do not disclose or use for any purpose. We may monitor all incoming and outgoing emails in line with current legislation. We have taken steps to ensure that this email and attachments are free from any virus, but it remains your responsibility to ensure that viruses do not adversely affect you. Cancer Research UK Registered in England and Wales Company Registered Number: 4325234. Registered Charity Number: 1089464 and Scotland SC041666 Registered Office Address: Angel Building, 407 St John Street, London EC1V 4AD.
