Mutagenesis kits for deletions

Frank O. Fackelmayer Frank at Fackelmayer.de
Tue Jun 12 06:14:11 EST 2001


We have often done it without any kit. That´s the method we used, it
works with any vector:


* let synthesize two primers that flank the region to be deleted. The
primers must face OUTWARD from the region to be deleted. The primers can
be designed like normal PCR primers, no need to do anything special (we
use 21-24mers). Order the primers 5´ phosphorylated!

* It is often a good idea to incorporate half a restriction site into
each primer´s 5´ region (then it is much easier to later identify the
positive clones). If you take a 6-base-cutter, the reading frame will
not be destroyed.

example:

3´-aaaaaaaaa//aaaaaaaaaaaaaaGGA                     
nnnnnnnnnnnn//nnnnnnnnnnnnnnnnnNNNNNNNNNNNNNNNNNNNNNnnnnnnnnnnnnnnn//nnnnnnnnnnnnn
nnnnnnnnnnnn//nnnnnnnnnnnnnnnnnNNNNNNNNNNNNNNNNNNNNNnnnnnnnnnnnnnnn//nnnnnnnnnnnnn
                                                    AGGaaaaaaaaaaaa//aaaaaaaaaaaaa-3´


would delete the N´s, while incorporating a BamHI at the junction.


* perform a PCR "around the plasmid", using a proofreading polymerase
(we use 7.5units of Pfu-Turbo) with the following conditions:

50ng template DNA
25pmole of each primer
2µl dNTP-mix (10mM each)
5-7.5 units of polymerase
and standard buffer supplied with the polymerase (including MgCl2 etc.)

in a 100µl assay.

Cycle:

95°C 5min preatreament

then 25-30 cycles
94°C 1 min
52°C 30 sec
72°C (length of the plasmid in kb)*2+1 min (e.g. 11min for 5kb plasmid)

then cool down to 4-10°C

* add 20 units DpnI directly to the PCR mix, mix, and digest for 1h at
37°C (will remove the template because it - but not the PCR product - is methylated)
* take a 10µl aliquot and run it on a gel. A PCR product of the expected
size should be evident. If it is not, we found it useless to continue.
Instead, optimize the PCR (eg. by adding 2%DMSO to the mix)
* purify DNA over a spin column, precipitate with EtOH and redissolve in
10µl of water
* add ligase buffer and ligase, and ligate overnight on the bench.
* transform bacteria. Use highly competent bacteria!
* pick clones for minipreps, and analyse for a.) the size and b.) the
presence of the incorporated restriction site. Presence of the
restriction site indicates the reading frame is ok. 
* sequence 2-4 positive clones to make sure they are ok

Hope this helps,
Frank




"M.P. Frosch, MD, PhD" wrote:
> 
> I'm looking for advice about choices among commercial mutagenesis kits that
> people have used for doing moderate sized deletions (20-50 bp).  I would
> like to do the mutagenesis in my own vector, but would reclone the DNA if it
> made an enormous difference.
> 
> Thanks for any advice.
> 
> Matthew Frosch
> --
> M.P. Frosch, MD, PhD
> 
> Center for Aging, Genetics and Neurodegeneration
> B114; Room 2700
> Massachusetts General Hospital
> 114 16th Street
> Charlestown, MA 02129-4404
> (617) 726-1265 (office)
> (617) 724-1480 (fax)




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