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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 01-23-2008, 02:29 AM
Felipe da Veiga Leprevost
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Default Gateway System




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."

Hi Koen,
What protocol are you following to enter the plataform? = Are you
making the BP reaction to clone your DNA into the entry vector?
Leprevost
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  #2  
Old 01-23-2008, 05:16 PM
| |Koen
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Default Gateway System

Hi Felipe,

no I am not using BP clonase to clone our gene of interest into the
pEntr vector. Instead, I want to subclone it from another non Gateway
construct that contains my gene of interest.

Koen

Felipe da Veiga Leprevost wrote:
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