2008/9/1 Artem Evdokimov <[EMAIL PROTECTED]>
> 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.
>
> Hello,
Artem is right in making this point of course, but why?
I think the number of moles of DNA molecule that you get is important. With
PCR you can get a huge number of moles, especially for a very intense 600bp
band. As you go up in size of product, the yield often becomes lower, and
it becomes more difficult to clone, but still, you'll probably have more
moles of insert compared to using restriction enzymes to cleave a fragment
out of a vector.
So when is using a TA cloning vector step justifiable? If you have a meagre
PCR product which you really can't clone any other way, TA cloning is the
most efficient with these low yields (for me).
Can high PCR yields inhibit restriction enzyme-based ligations? Yes, if you
don't cleave these products to completion. If you have PCR products where a
significant proportion are not cleaved properly, the non-cleaved molecules
will have an inhibitory effect on your reaction- one can imagine that
they'll mop up a large proportion of your vector and make it unusable. So,
if you have a huge PCR product, try cleaving 10-30% of the entire reaction
either 4 hours or over night to make sure it's fully cleaved. (Again, as
Artem says, choose your buffer carefully here).
As a final piece of advice, make sure you have a copy of "Molecular Cloning,
a Laboratory Guide" (Cold Sring Harbor Press) nearby.
http://books.google.fr/books?id=YTxKwWUiBeUC&dq=molecular+cloning+a+lab+manual&pg=PP1&ots=FVO87K4uEt&sig=xTYcKpFP45dBqwsfRWZ0UXR0wb8&hl=en&sa=X&oi=book_result&resnum=1&ct=result
...You will probably have a recent or not-so-recent edition (A.K.A.
"Maniatis" for previous editions IIRC) in a University Library nearby. I
recently frog-marched a technician here to read it when he was having
cloning problems. After persuading him that the PEG-additive protocol might
be helpful to help him with a particularly nasty cloning problem, he tried
it. As he said, the results were "miraculous". (I have no affiliation to
CSHL press nor the authors BTW)
Good luck,
Mark
>
>
> 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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--
Mark BROOKS
Telephone: 0169157968
Fax: 0169853715
Institut de Biochmie et de Biophysique Moleculaire et Cellulaire
UMR8619 - Bât 430 - Université de Paris-Sud
91405 Orsay CEDEX
Skype: markabrooks
