TA cloning and mutation - (Nov/17/2006 )

HI all
I'm tring to clone a 3,1 Kb PCR fragment (consisting mostly of 3'UTR) of my gene of interest in an Invitrogen TA cloning vector containing GFP at th 5'.
I'm using High fidelity taq polimerasy (both from eppendorf and from initrogen - taq platinum-) to amplify the target and UV free kit to purify the PCR products.
After very good transformation, I sequence the plasmid to chek the insert sequence but I keep finding single point mutation (no less then 10mutation in 3 Kb of insert).
I need a plasmid with the right sequence but i can find it.
is anyone that can tell me why?
thank you all
barbik

Are you 100% sure about your original sequence? Are they always the same mutations? (this could give indication that they are indeed PCR-induced).

Apart from this: try using a pure proofreading enzyme istead of a mix (high fidelity taq is a mixture of taq and a proofreading enzyme, resulting in less mutations than taq on its own, but more than a pure proofreading enzyme). For this you will need to blunt clone, or have taq added afterwards (for extra A adition).

One question
Are all the point mutation in your various clones in different positions? What I am trying to say is, are these "mutations" true mutations and not an actual difference between the sequence that you have and the actual physical gene you have in your tube.

If these mutations are real, with each clone having its own unique set of mutations,
I would suggest changing the polymerase to a proper high fidelity polymerase like KOD, rather then a mutated taq.

I presume that your template is genomic DNA. If it is cDNA from Reverse transcriptase PCR, there migh have been an error in that reaction's buffer mix.. causing more then the usual number of mutations.

EDIT: Well looks like I have the same advice as vairus

Are the mutations in the same place if you resequence? I had a similar problem, but it turned out to just be the sequencing program miscalling bases, when I resequenced these "mutations" had moved.