directional cloning??

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kabuto
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directional cloning??

Post by kabuto » Sat Nov 11, 2006 10:14 am

what is this?
can someone explain in simple terms

Methal
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Post by Methal » Sun Nov 12, 2006 1:15 am

Directional Cloning is when DNA is inserted and vector molecules are digested with two different restriction enzymes to create noncomplementary sticky ends at either end of each restriction fragment. This allows the insert to be ligated to the vector in a specific orientation and prevent the vector from self-ligation.

And Yes, i totally googled that...

and no, it doesnt make a lot of sense to me either.

basically I would say its more specific cloning, such as induced mitosis/meiosis.
That'll do donkey, That'll do.

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canalon
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Post by canalon » Sun Nov 12, 2006 9:07 pm

If you have a DNA fragment that have the same restriction site (a) on both ends it can be inserted (cloned) in both direction:

vector-a a-fragment-a a-vector

OR

vector-a a-tnemgarf-a a-vector

Which may no be practical in case you want the expression of you fragment. If you make a double digestion (of vector and fragment) with 2 different enzymes, thus yielding defined and diffrent sticky ends (a and b)on each side you wil be sure to get that

vector-a a-fragment-b b-vector

AND NOT

vector-a b-tnemgarf-a b-vector
Patrick

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any proof. (Ashley Montague)

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MrMistery
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Post by MrMistery » Mon Nov 13, 2006 6:23 pm

methal, your explanation maked sense to me
and thanks patrick for clearing that up
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

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