Alternatively, you could skip troubleshooting digestion/ligation/etc.
and use a kit based on site-specific recombination, like the In-Fusion
kit that Clontech sells. (I don't have any financial interest here--I'm
just a graduate student, but I've had good results using it.) The kit
is not without its own downsides--it's a bit pricier than traditional
cloning, you'd have to design another set of specific primers, and you
have to be very careful about the vector:insert ratios you use.
Good luck,
Brian
Artem Evdokimov wrote:
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
------------------------------------------------------------------------
*From:* CCP4 bulletin board [mailto:[EMAIL PROTECTED] *On Behalf
Of *vijay srivastava
*Sent:* Monday, September 01, 2008 3:06 AM
*To:* [email protected]
*Subject:* [ccp4bb] regarding cloning
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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