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remember DpnI digestion doesn't work at all on DNA isolated from non-bacterial sources or bacteria lack Dam methylase, such as JM110 strain. and will result in a high background of wild types colonies with few or no mutants. Padayatti On Mon, Sep 30, 2013 at 12:54 PM, Matthias Haffke <[log in to unmask]>wrote: > Hi! > > I would like to suggest a different site-directed mutagenesis (STM) > protocol than the ones, which are commercially available or commonly used. > > We constantly use what we call a "self-SLIC" STM protocol. Simply follow > the SLIC method for cloning as described by Li & Elledge (*Nat Methods.<http://www.ncbi.nlm.nih.gov/pubmed/?term=harnessing+SLIC#>2007 Mar;4(3):251-6. > *), by using two standard primers (forward & reverse), which both include > the mutation(s) of interest in the homology region needed for SLIC. Amplify > the whole plasmid of interest with these primers, do the T4 DNA treatment, > DpnI digest to remove the template, anneal and you will have clones without > any bias in your mutated region. This is because the T4 DNA polymerase > treatment removes the newly synthesized strand in your mutated region (wich > is your SLIC homology region) to have two 5' overhangs for annealing. These > 5' overhangs of your linear PCR product are ONLY defined by the primers you > use and will be independent of the actual PCR amplification cycles. We have > done STMs with >12bp of mutations in a single reaction without any problems > using "self-SLIC" STM. > > This figure will hopefully help you to understand the rational behind the > "self-SLIC" STM: > > > > > > Of course, this requires primers of good quality, but I have never seen > before that we received a mix of primers. We order ours from Invitrogen and > use standard desalted samples, 25 nmol synthesis scale. > > > > *The reference for the Li & Elledge paper: > > Li & Elledge (Nat Methods.<http://www.ncbi.nlm.nih.gov/pubmed/?term=harnessing+SLIC#>2007 Mar;4(3):251-6. ) > * > > > Best, > > Matthias > > > ------------------------------ > Date: Mon, 30 Sep 2013 17:19:19 +0200 > From: [log in to unmask] > Subject: [ccp4bb] Off-topic: Site-directed saturation mutagenesis trouble > To: [log in to unmask] > > > Dear CCP4ers, > Sorry for the off-topic email. We are currently experiencing a bit of > trouble with our site-directed mutagenesis and hope that some of you have > experience with this. > Our sequencing chromatgograms suggest that the primers are not of good > quality as the bases are not represented euqally or that the PCR did not > lead to good incorporatoion of the bases - see attatched PDF. Is this sort > of chromatogram the best we can expect? So my questions are: Where do you > order primers? Do you do any special synthesis or are there important > requirements? What kind of mutagenesis protocol would you use? What quality > of randomisation can one expect? > We would be very grateful for any advice you can offer us. > Thanks in advance. > All the best, > Hazel > M.Sc. Hazel Fuchs > Ph.D. Student of Biochemistry > Department of Cellular Chemistry > Medical School Hanover > Germany > > -- PSP