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gateway cloning

gateway cloning - Protocols and Methods Forum

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


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  #1  
Old 01-07-2009, 09:51 AM
| |Koen
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Default gateway cloning



Hi,

we are currently using the Invitrogen Gateway system to make
expression vectors for our genes of interest. We do so by using
pDonr223 and our PCR amplified cDNA containing the recombination
sites. For some genes this works well but for others we have
difficulty in getting colonies containing the cDNA of interest. Our
problem does not seem to be due to the length of the cDNA as we have
succesfully made Entry clones containing cDNAs up to 5000 bp without
any problem. Any ideas about the reseans for the varying efficiency of
generating Entry vectors using the pDonr and BP-clonase 2 and the PCR
amplified fragment containing recombination sites?

Koen
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  #2  
Old 01-09-2009, 09:38 AM
StewJW
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Default gateway cloning

Hi Koen,

I don't claim to be an expert in this area, but have you tried
linearising your attB expression clones as described in the
Invitrogen manual. Also, have you purified the PCR product to remove
attB primers and any attB primer-dimers. Invitrogen also recommends
not to use phenol/chloroform extraction clean up methods. Like you say
its unlikely to be a size issue which is more of a problem with making
entry vectors using TA cloning methods where the upper limit is around
5kb. There is also something else nagging me about problems witrh
certain sequences, which is unpublished but I can't remember what it
is may be someone else can help here.

Best,
Stewart
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  #3  
Old 01-09-2009, 09:41 AM
StewJW
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Default gateway cloning

I should also add have you performed control reactions to check your
reagents, occasionally there are problems with the clonase mixture,
and Invitrogen provides controls in their kits.
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  #4  
Old 09-18-2010, 07:36 AM
Pipette Filler
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Default Re: gateway cloning

Hi Stewart,

I realize these posts have been made quite a long time ago, but I am still hopeful that I may garner a response. I have spent much time with Gateway cloning and subsequent troubleshooting to no avail. I am very interested about your comment as to certain sequences proving difficult to clone, and am wondering if you have any more information regarding that unpublished bit of info....

Laura
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