perhaps the only real way to find out is to sequence 20-50 different clones of the final library and see if you get decent randomisation.
Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 1 Oct 2013, at 11:00, Hazel F. wrote: > Dear CCP4ers, > > Thank-you very much for your many and fast answers! But I suspect you > misunderstood my question- I am having problems with site-directed saturation > mutagenesis- I try to incorporate a multitude of mutations in to a library. > So I get mutations, but not correct represention of all the bases I would > like to incorporate at the relevant position. So for example in the > sequencing I attatched I wished to see N (G, A, C and T) at the first > position but the bases are not equally present. > > Can someone give me advice on how to get better incorporation of all bases? > Or is this quality of sequencing (see attatchment) normal? > > Thanks in advance! > > All the best, > > Hazel > > M.Sc. Hazel Fuchs > Ph.D. Student of Biochemistry > Department of Cellular Chemistry > Medical School Hanover > Germany > > Von: CCP4 bulletin board [mailto:[email protected]] Im Auftrag von Hazel > F. > Gesendet: Montag, 30. September 2013 17:19 > An: [email protected] > Betreff: [ccp4bb] Off-topic: Site-directed saturation mutagenesis trouble > > 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 > > <LibrarySeq.pdf>
