J_MCLAUGHLIN@hvrford.bitnet (01/06/90)
We are involved in making single base mutations in ribosomal RNA and then
examinining how the mutations influence function. To make the mutations
we have utilized or should I say attempted to utilize the Bio-Rad Muta-Gene
kit. We continually have problems- I have checked all the reagents, my
oligos and we still cannot get mutations. So, if anyone utilizing this
method has some suggestions please let me know. Also, if you have any info
on the E. coli strain- whether the dut ung efficiency inserting uracils
is affected over time. Or, if anyone has some other "so-called" easy method
to make mutations such as the T7 procedure from US Biochemical tell me
PLEASE. The last thing I want to do is spend all of my time making mutations
we want to see what they do!
Thanks
J_MCLAUGHLIN@HVRFORDdrsmith@silver.ucs.indiana.edu (drew smith) (01/06/90)
In article <9001051847.AA22125@genbank.bio.net> J_MCLAUGHLIN@hvrford.bitnet writes: >We are involved in making single base mutations in ribosomal RNA and then >examinining how the mutations influence function. To make the mutations >we have utilized or should I say attempted to utilize the Bio-Rad Muta-Gene >kit. We continually have problems- I have checked all the reagents, my >oligos and we still cannot get mutations. So, if anyone utilizing this >method has some suggestions please let me know. Also, if you have any info >on the E. coli strain- whether the dut ung efficiency inserting uracils >is affected over time. Or, if anyone has some other "so-called" easy method >to make mutations such as the T7 procedure from US Biochemical tell me >PLEASE. The last thing I want to do is spend all of my time making mutations >we want to see what they do! > > Thanks > > J_MCLAUGHLIN@HVRFORD It would be helpful if you included more specifics - there are plenty of things that could be wrong. I suggest these controls, if you have not done them already: 1) Assay for primer extension - there are two ways to do this, and both should be used. The first is to look for the generation of ccc and nicked DNA by electrophoresis on an agarose gel containing 2ug/ml EtBr. The second is to measure the transformation efficiency of your extension reaction against template-only, and your no primer control. If the efficiency of the extension reaction is not at least 10X increased over the controls, you are in trouble. 2) The efficiency of uracil incorporation can be monitored by the transformation (or transfection, depending on your vector) efficiency of your template into an ung+, dut+ host. Efficiency should be reduced at least 2 orders of magnitude. Most probably you are not getting good primer extension and ligation. - Drew Smith Dept. of Biology, Indiana University