Hi,

 

First of all – I am curious why did you decide to put in an extra step (the T/A cloning into an intermediate vector)?  You can happily digest your PCR product with NheI/BamHI, clean up and ligate into the appropriately digested pET-23a(+). If you have issues, you should definitely try this.

 

Now, since you do have an intermediate step – did you verify that everything was OK after havig subcloned your insert into whatever vector you’re using? Did you sequence the insert and most importantly did the sequencing confirm the nature of the linker regions?

 

The enzyme pair that you chose has a slight issue with digestion buffer – most people would choose NEB buffer 2 (since buffer 3 is bad for Nhe) where Bam still has ‘100% activity’ – however, in buffer 2 you can have star activity of the Bam due to the somewhat lower salt concentration (50 mM instead of the optimum 100 mM). It’s not impossible to imagine that you have issues with digestion. This can be easily avoided by sequential digestion although of course it’s slightly more work (but if you cut out the T/A cloning step that’s actually still faster).

 

So, in conclusion the most likely issue is digesiton (probably of the pET vector, to be more specific). Next likely issue could be ligation – make sure that you base your ligation ratio on the gel intensity of the bands as well as on the OD260 of your DNA. Faulty primers are not likely to be an issue since you seem to be able to restrict your insert out of the intermediate vector.

 

Please note that you can often use SpeI or XbaI instead of Nhe since they have compatible sticky ends. Clearly this depends on the vector you’re working with and I am too lazy to look up pET23 polylinker.

 

Artem

 

Hi, I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning.  The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. I was succesful  in subcloning (T/A vector) and getting my insert at 1.2kb after  double digestion and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is 1:3,1:2,getting the colony after the transformation but some how  when i used to confirm my clone through double digestion i am not getting my insert at the correct position.Some time in the gel only the size of the vector was there.

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