-----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-----
