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Not understanding "rendered flush" concerning vectors

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Not understanding "rendered flush" concerning vectors

Postby kunito » Wed Sep 21, 2011 9:31 pm

I'm helping to do translations of an article concerning binary agrobacterium vector for plant transformations and I'm having difficulty with this term "rendered flush." What does it mean in this sentence, "The Dde framgments were "rendered flush" with Klenow polymerase?" I have been searching for this term in relation to the field of molecular biology but not getting answers or just getting info about html rendering or something like that.

I would appreciate any help. Thank you.
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Postby canalon » Thu Sep 22, 2011 2:14 am

That the Klenow fragment of the taq was used to make the fragments flush by filling the gaps created by the restriction enzyme, i.e. cut with a RE then made the DNA fragment blunt ended.
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Postby merv » Sun Oct 02, 2011 3:03 pm

That is more or less correct; cutting enzymes usually leave the DNA helix cut at staggered lengths, so they have 'sticky' (cohesive) ends. Although these often make DNA ligation (joining) easier, this only applies if the ends to be joined are complementary to each other- as this often not the case, it is necessary to blunt the ends- if Klenow fragment of DNA polymerase is used (E.coli DNA polymerase I lacking the proofreading component, the most frequently used enzyme for this process), the ends can be blunted- if sufficient nucleotides are included, the single strand will be converted into double strand, if not the single strand will be degraded. Although this process will not be perfect (i.e. many partially filled or degraded ends will persist), the DNA ligase will only join the blunt ends into the blunt ended vector. This is a more difficult cloning reaction than a cohesive-end cloning reaction.
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Re:

Postby merv » Sun Oct 02, 2011 3:12 pm

canalon wrote:That the Klenow fragment of the taq was used to make the fragments flush by filling the gaps created by the restriction enzyme, i.e. cut with a RE then made the DNA fragment blunt ended.


obviously not taq but DNA pol I Klenow fragment there. Although Taq pol is as you probably know also used to render staggered ends blunt, again requires dNTP's of course.
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Re: Re:

Postby canalon » Mon Oct 03, 2011 12:38 am

merv wrote:
canalon wrote:That the Klenow fragment of the taq was used to make the fragments flush by filling the gaps created by the restriction enzyme, i.e. cut with a RE then made the DNA fragment blunt ended.


obviously not taq but DNA pol I Klenow fragment there. Although Taq pol is as you probably know also used to render staggered ends blunt, again requires dNTP's of course.


Yeah my bad for that. Thanks for pointing it out.
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