Fixing SNPs after cloning
Posted 23 January 2012 - 06:25 PM
Last time when I cloned a short gene which is about 1kb everything was completely fine the sequence was exactly the same as I wanted it to be, however as soon as I go for a bigger insertions I immediately get nucleotide polymorphisms. So what is the common strategy to overcome this problem? Thanks for advices in advance!
Posted 23 January 2012 - 07:48 PM
Posted 24 January 2012 - 03:10 PM
Posted 24 January 2012 - 06:30 PM
The long range Roche enzyme makes only 3x less errors of normal taq, where as high fidelity enzymes like Phusion have 50x less errors than taq.
So I think it is possible that you are getting a bunch of errors in your PCR and then picking them up in your different clones. I've used Phusion for products as large as 4.5kb so I think you might be ok using it for your larger fragments too.
Alternatively, if the errors are occurring in your bacteria, perhaps it has to do with the strain you are using? Can somebody else help on this one?
Posted 24 January 2012 - 09:16 PM
Posted 25 January 2012 - 12:39 PM