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:[log in to unmask]] Im Auftrag von Hazel F.
> Gesendet: Montag, 30. September 2013 17:19
> An: [log in to unmask]
> 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>
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