site-directed mutagenesis

[Uofc]

Hi,
I want to mutate the active site of my protein which is Histidine. can anyone suggest if I can do it to asparagine.

[relaxin]

I think it is better to change it into something that is simple and carries no positive charge, such as alanine or glycine.

[CrowSan]

For the mutagenesis I would recommend getting a kit if you have not done it before (it is often cheaper to put a kit together yourself later by buying e.g. Dpn1 and taq pol seperately).
I have used QuickChange mutagenisis kits before with good results (not affiliated) although many other kits are available.
The base change from His to Aln is simple (1 base) and it will be the first base of the His codon (i.e. if His = CAT then change it to AAT = Asn, if His = CAC the change to AAC = Asn).

You will need your gene in a plasmid and you will need bacteria.

[CrowSan]

May have mis-read your question. In short people have mutated His to Asn before, i.e.
http://www.jbc.org/content/261/7/3363.full.pdf

[morkfromork]

For the mutagenesis I would recommend getting a kit if you have not done it before (it is often cheaper to put a kit together yourself later by buying e.g. Dpn1 and taq pol seperately).
I have used QuickChange mutagenisis kits before with good results (not affiliated) although many other kits are available.
The base change from His to Aln is simple (1 base) and it will be the first base of the His codon (i.e. if His = CAT then change it to AAT = Asn, if His = CAC the change to AAC = Asn).

You will need your gene in a plasmid and you will need bacteria.

I can also recommend QuickChange, it is efficient and straight-forward. I learned SDM by doing 4 primer recombination PCR which requires a number of steps and can be fiddley. The kit works in one day (plus the time needed to miniprep the bacteria).