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gateway system

gateway system - Protocols and Methods Forum

gateway system - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 01-22-2008, 04:06 PM
| |Koen
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Dear all,

I want to use the gateway system of invitrogen to minimize the
workload of subcloning our cDNAs in various expression vectors. Before
using the system I have to clone our cDNAs in an appropriate pEntr
vector. I used pEntr1a for this but I do not get any colonies on
kanamycin plates using homemade DH5alpha or Invitrogen Top10 cells. I
did remove the ccdB sequence before putting in our cDNA. I also tested
cutting out the ccdB sequence, religating the empty vector and
transforming bacteria with this. I get only a very limited amount of
colonies from transformed Top10 cells and none on our own competent
DH5alpha.

can anyone tell me what I did wrong?

Koen
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Old 01-23-2008, 01:10 AM
DK
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In article <[Only registered users see links. ]>, "| |Koen" <[Only registered users see links. ]> wrote:

Have you considered the fact that the cloning artifacts that it
inevitably reproduces (please correct me if I am wrong) may
skew your results significantly?

I've seen more than one example of just a single "strange"
amino acid significantly affecting protein's properties.

Before

So, if I understand correctly, you are not getting ligation for cloning
into pEntry clone to work? Is this right? If yes, then, unfortunately,
there is almost an endless list of possibilities and, unless you have
done all the usual ligation troubleshooting controls and can list
them, there is no way for others to know what went wrong.

If you are talking about recombination from pEntry into
other vectors not working then, since this would be using a kit for
which very little concrete information is available, it's hard to tell
where problems are. The simplest is that you have dirty miniprep
that carries over some inhibitors into the reaction. Try cleaning
up your vector.

DK
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