Results of T-Vector Poll

cjharrod at waikato.ac.nz cjharrod at waikato.ac.nz
Mon May 30 08:47:48 EST 1994


Netters!

Several weeks ago I proposed a Poll on what sort of vector people use for
cloning PCR products.  The results were:

Invitrogen TA Vector............3
Promega pGEM-T..................1
Marchuk Method*.................1

* Marchuk et al (1991) NAR 19: 1154.  The method involves blunt end cutting
  a vector, then incubating vector with dTTP and Taq.  Any vector can be
  made into a TA-Vector.

A clear winner - the most expensive vector on the market.

The responses are summarised below, for your reading pleasure:

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Timo Hiltune (Timo.Hiltunen at csc.fi) wrote:

Hi!

Invitrogen's TA Cloning Kit (version I) worked fine with me without
any major modifications and even with lower amounts than suggested in
the instructions.

Right now I'm working with Promega's pGEM-T-Kit, and it doesn't work when
I do it with the instructions. With modifications of transformation and 
length of ligation, I am trying to proceed now, because this kit is
cheaper than InVitrogen's.

BTW, this way of setting up a poll may be an effective way of getting
information when it is needed urgently. I hope you will summarize, anyway.

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Claudia Sutton (cas9 at cornell.edu) wrote:

I have had usually good luck with invitrogen TA kit, but want to try a
homemade kit--the cost is significant.  By the way, I never do a long final
extension in my amplifications.  I like having amp and kan resistance in
the Invitrogen vector so that you can avoid precursor plasmid contamination
when pcr-ing from subclones.

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Melissa (sistrunk at bioc.rice.edu) wrote:

My two cents worth:

I used the Invitrogen TA Cloning kit to clone 6 different PCR products.  I
needed them each to clone into the vector into one particular way because I
wanted to cut the inserts out with certain restriction enzymes from the
polylinker to further subclone them.  However, three of the clones only
inserted in the wrong direction, based on sequencing of >15 clones each. 
So, be careful if orientation matters to you.

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Alan (pdzah at pdn1.gene.nottingham.ac.uk) wrote:

Re PCR cloning
                      I always use Promega T-vector with consistently 
good results even when ligating straight from LMP agarose.
I always make my own competent cells(JM109) and use them the 
same day. This normally gives 20-30 colonies of which 30-50% 
contain the sequence of interest. Desired inserts are found in 
both blue and white colonies.
Hope this helps your survey,
                                             Cheers 
                                               Alan

I think I omitted to say that the vector is actually called pGEM-T.

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Ed Rybicki (ed at micro.uct.ac.za) wrote:

As per Marchuk et al., exactly.  Good results on a variety of amplimers.

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And there you have it.  Hope this was enlightening!



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