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Gene insertion without desired unique restriction sites


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#1 SOUMYA RAO

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Posted 27 September 2009 - 10:32 AM

Hello friends,

while working on a cloning experiment i came across a problem.
I have to clone a gene into a binary vector that has been modified and contains only two restriction sites-NcoI and XbaI.
I need to use only this one to clone some genes that have either of the two or both the sites internally.
How can i use the same vector,ligate my insert into it and prevent my gene from being chopped off on restriction digestion with the above enzymes?

#2 Warren

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Posted 27 September 2009 - 12:43 PM

Hello friends,

while working on a cloning experiment i came across a problem.
I have to clone a gene into a binary vector that has been modified and contains only two restriction sites-NcoI and XbaI.
I need to use only this one to clone some genes that have either of the two or both the sites internally.
How can i use the same vector,ligate my insert into it and prevent my gene from being chopped off on restriction digestion with the above enzymes?


Break out your New England Biolabs catalog and look up compatible cohesive ends. For example, I know SpeI and NheI (both common enzymes most people have) are compatible with XbaI -- so if your gene doesn't contain these sites, then those could be used. The compatible ends for NcoI are less common enzymes, but then again, you could use any enzyme and blunt this end, and blunt the NcoI site; with a single cohesive end, the cloning is still pretty easy. Warren..

#3 perneseblue

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Posted 28 September 2009 - 12:02 AM

Should you be unable to use restriction enzymes that have compatible cohesive ends (ei NheI, SpeI), you could consider changing the restriction site in your binary vector to something more suitable. You can

1- clone in a synthetic oligo linker into your vector. This linker can be designed to delete the restriction site that it goes into.

2- you can PCR amplify your vector with primers that contain your desired restriction sites.
May your PCR products be long, your protocols short and your boss on holiday

#4 SOUMYA RAO

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Posted 28 September 2009 - 10:02 AM

Should you be unable to use restriction enzymes that have compatible cohesive ends (ei NheI, SpeI), you could consider changing the restriction site in your binary vector to something more suitable. You can

1- clone in a synthetic oligo linker into your vector. This linker can be designed to delete the restriction site that it goes into.

2- you can PCR amplify your vector with primers that contain your desired restriction sites.




Thanks a lot,
I will surely consider this if don't get any compatible sites for NcoI.
Soumya.




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