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religate the vector - need good protocol!


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4 replies to this topic

#1 lna

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Posted 17 October 2009 - 12:20 AM

Hi everyone!
though usually in the molecular cloning it is needed to avoid the self-ligation of the vector, now I really need to do it!
So, I've got the vector, cut by BclI and treated with T4 pol. Now I need to do this blunt self-religation to get the circular vector. Can anyone give me a link to the best protocol? Because all I've found are the protocols for the vector-insert ligations. I use the Invitrogen T4 ligase, if matters.
Thanks!!

#2 Michaelro

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Posted 17 October 2009 - 12:31 AM

Hi
It is a regular ligation only without insert.
It should be easy.
After purifying your fragment from the T4 polymerase reaction, put some like 50ng into the ligation reaction which contains all the components except the insert. And follow the regular ligation protocol (in your case of Invitrogen).
Good Luck

#3 lna

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Posted 17 October 2009 - 12:46 AM

Hi
It is a regular ligation only without insert.
It should be easy.
After purifying your fragment from the T4 polymerase reaction, put some like 50ng into the ligation reaction which contains all the components except the insert. And follow the regular ligation protocol (in your case of Invitrogen).
Good Luck


thank you for quick response!
well, in fact the point is that I've already did it. and got some strange tiny colonies. I'm afraid that with the standard protocols I will get multiple religation products.. for Invitrogen there are two protocols provided: one for cohesive ands and another for the blunt.

#4 phage434

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Posted 17 October 2009 - 07:43 AM

Low concentration of the DNA will favor recircularization rather than multimers. But multimers are not usually a problem, since multiple plasmid origins are often non-functional.

#5 swanny

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Posted 18 October 2009 - 03:39 PM

I am presuming you are T4 pol-treating the vector for a LIC/SLIC cloning reaction, or at least your reaction mix does not have dNTPs. If you've just been filling in the sticky ends, proceed to a blunt-end ligation.

After T4 pol treatment, you should have long ssDNA overhangs. Try adding some dNTPs to the T4 pol reaction mix (plus a DNA pol if you've denatured the T4 pol). This will make blunt-ended dsDNA, on which you can do a standard blunt-end T4 ligase reaction.
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