promoter mutagenesis question

[bford]

Wolfgang;

In a previous life I had a similar problem with a
prok expression vector. If you have not already done the hard 
cloning part, take some time to do this right. Just filling in the XHO 
site is one option, but you might want some useful site there after all.
Also, your vector grows by 4 bp and two restriction sites. Is that a 
good thing? 

How about this.....
Based on certain assumptions about the XHO I location....
Design the MCS/promoter site you should REALLY have, then cut out the
MCS that exists, clone back in the new one (two oligos). In this way
you can add/delete more than the XhoI site, and adjust spacings etc if 
you need. In the ligation mix, you don't need to seperate or anything.
Overload a bit with the oligos.
You'll get parentals, recombinants, and bastards, but you just need to 
screen a handful to get the right construct. Not so bad.

Or this:
Rather than Klenow, treat with a nuclease (eg Mugn bean) or exo/endo + 
polymerase which can eat free ends in the absence of nucleotides (eg 
T4). Let it run to completion, then add pol/nucleotide mix to fill in 
scruffy ends. 
You'll need to characterize the product carefully, but I have used this 
approach a couple of times to reduce promoter-ATG spacing. OTOH, Mung 
bean is a bit unpredictable.


It's more work at the beginning, but in the long run, you get a vector 
you can actually use. 

B.