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Problem with TA cloning - (Aug/17/2007 )

We have been cloning genes recently using TA vector and TOP10 cells from invitrogen,

after ligation/transformation we can confirm presence of the insert by PCR and sequencing but cant cut the gene out with the engineered restriction sites (NotI and BamHI), anyone else experienced this or got any reasons as to why ??

we have resorted to PCRing from TA using m13 primers and cutting that product which works fine

is it just that invitrogen are rubbish ?



When you sequenced your insert were the restriction sites intact? There can be mutations in those sites or maybe the primers you ordered don't have the correct sequence for those enzymes...

Are you sure your restriction enzymes are working and that your conditions for digestion are the best? Test them with some other construct to check this.