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~~~~~HElp!!! Research LR reaction!!!*******

Discussion in 'Pre-Medical - MD' started by greenteapudding, Apr 12, 2007.

  1. greenteapudding

    7+ Year Member

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    It's probably a stupid question...please help! I'm working on converting a cloning vector to a gateway destination vector for LR reaction!

    I'm trying to insert a invitrogen LR reaction attR site into a plasmid by restriction enzyme (blunt ended), but i dunno which reading frame i should be using for the fragment. There are three cassettes of the attR fragment to choose. But I have trouble understanding how to identify the reading frame of the plasmid, and how to keep the reading frame by choosing the right cassette...

    Here's the kit info and manual:
    https://catalog.invitrogen.com/index...geid%3D102 87

    Any help would be appreciated...Thanks!
     
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  3. GreenShirt

    10+ Year Member

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    We work with this system all the time in my lab. It's really efficient. It depends on what complementary att sites you have on your entry vector, the manual should have a list of which sites are compatible w/ which ones, although the info is buried in a lot of text so you might have to look more carefully. Your ORF is going to start translating at the start codon of your insert, so the frame of the att sites doesn't matter.
     

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