Site mutagenesis on 11kb plasmid
-optimized annealing temp (initially started with gradient pcr)
-DpnI digestion carried out (checked via gel and obtained a large smear)
-Transformation (Kanamycin resistance):
1. original plasmid gave colonies in DH5alapha (no problem with size)
2. pcr products did not gives colonies in DH5alpha
3. pcr products gave colonies in NEB10beta; but sequencing showed no successful mutagenesis.
seems like DH5alpha can transform large circular DNA. maybe linear large dna is more difficult?
should i redesign the site mutagenesis primer (however current primer has no problems annealing)?
In my experience site directed mutagenesis is sometimes inefficient or does not work at all (has to do with sequence composition). I have developed ways to overcome this but since I now have a company for cloning services I can only offer to do this for you as a paid service. The cost should be $315-$450 depend on the sequence. If interested feel free to contact me through our website http://www.customdnaconstructs.com/
Best,
Nahum
http://www.customdnaconstructs.com/
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