PCR mutagenesis-strange results

Frederik Boernke boernke at nospam.ipk-gatersleben.de
Mon Nov 22 15:17:30 EST 1999


Hi all,

I did some PCR mutagenesis on my gene using Stratagene's quick
change kit. Everything seemed ok so far, since sequencing of the
respective region confirmed the nucleotide exchange. However, when
looking at the oligoes used I realized a mistake at the 3' end which
actually should introduce a frame shift into my sequence. 
The last bases of the oligo are: GAT TTG G but my sequence is GAT TGG.
The oligos where HPLC purified so they should be alright. Sequencing of
several independent mutagenized clones revealed no frame shift, the
additional T disappeared. How can this happen? Any ideas?

Ricky 
*******************************************************************

Frederik Boernke
Research Group of  Molecular Plant Physiology
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel.  039482 -5 321
Fax. 039482 -5 515
http://www.ipk-gatersleben.de




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