Site-specific nucleotide base mutagenesis

drew smith drsmith at silver.ucs.indiana.edu
Fri Jan 5 17:01:39 EST 1990


In article <9001051847.AA22125 at genbank.bio.net> J_MCLAUGHLIN at 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 at 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



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