J_MCLAUGHLIN@hvrford.bitnet (01/10/90)
Thanks to people for responding to my first message. I'm new to e-mail so
I wasn't sure exactly what to write. I'm also new to molecular biology in
the sense my degrees are in plant physiology and have been here at Haverford
for 2 years. Everyone asked for more specifics so here goes- I have cloned
a 1050 bp piece from ribosomal RNA of E.coli into M13mp19. Single strand mp19
with the piece is inoculated with the dut- ung- E. coli strain CJ236. SS is
then extracted, purified with PEG and phenol chloroform. A 17 b primer with
a single base mutation (in center) is annealed to the template after being
phosphorylated with T4 polynucleotide kinase. The actual rxn:
200 ng uracil DNA
2-3 pmol oligo
1 ul 10x annealing buffer (20mM Tris 7.4, 2mM MgCl2, 50mM Na
Cl.
70 degrees, cool till 30 degrees. Then for copying of the strand we use T4 DNA
ligase, T4 DNA polymerase, and T4 Gene protein. Synthesis buffer contains
.4mM each dNTP, .75mM ATP, Tris, MgCl2, and DTT. I run 1% agarose gels of the
DNA both before and after copying and it has indicated copying did occur. I
then transfected into E. coli strain XL1Blue dut+ung+. I get lots of plaques
but no mutations so lets here what everyone thinks.drsmith@silver.ucs.indiana.edu (drew smith) (01/10/90)
In article <9001092026.AA25497@genbank.bio.net> J_MCLAUGHLIN@hvrford.bitnet writes: >I run 1% agarose gels of the >DNA both before and after copying and it has indicated copying did occur. I >then transfected into E. coli strain XL1Blue dut+ung+. I get lots of plaques >but no mutations so lets here what everyone thinks. The important control is the mock reaction with no primer: you could be getting priming from small RNA contaminating your DNA prep. This is one way you can get lots of DNA synthesis, but no mutants. You might also want to try a polymerase other than T4. I've always found its lack of processivity to be a problem, and I don't know how much the gene 33 protein will help. Another approach to the processivity problem would be to clone into a smaller phagemid vector. Finally, sometimes you just happen to hit a repair hot spot. If all else fails, clone your gene backward and try again. - Drew Smith Dept. of Biology, Indiana University