Anything to say about invitrogens Gateway system?

David Micklem dmicklem at cmgm.nospam.invalid
Wed Dec 19 19:01:18 EST 2001


In article <9vqij4$h9s$1 at news.net.uni-c.dk>, Morten Lindow
<lindow at molpharm.dk> wrote:

>Hi fellow biologists,
>
>I am about to start a major protein expression project. I am going to make a
>lot of constructs with different tags and in different expressions systems.
>Hence I am considering using the Gateway system from Invitrogen.
>
>Can of you say something about it? Good and bad sides?
>Is it more or less reliable than traditional restriction-ligation
>cloning(e.g. how often do you get no colonies on your plates)?  What about
>the extra aminoacids that are introduced between your protein and a given
>tag?
>
>Best regards,
>
>Morten Lindow
>Univ Copenhagen

Hei Morten

I've used it quite a bit, and find that it is extremely reliable.
Almost as great as their TOPO-TA system for cloning PCR products.

As with TOPO-TA, you can scale the reactions down to make the kit go
further, although you will need to provide the extra competent cells
yourself: I've been doing 2ul reactions (1ul PCR product + 1ul of a mix
of 2:1:2 BP buffer:pDONR:BPenzyme). Then transform cells with all of
the reaction. (The kit protocol says to use 20ul reactions, but only
use 1ul in the transformation, so you end up throwing away 19ul....)

At the proteinase K step, adding 2ul of a 1/10 dilution of the
Proteinase K in TE has been working for me.

God jul,

David

-- 
D.R. Micklem,              Time flies like an arrow... 
Dept. of Anatomy,          Fruit flies like a banana.
Cambridge University,      Email:dmicklem at cmgm.stanford.edu 
Cambridge, UK              Phone: +44 (1223) 333776
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