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Ligating 2 Linear fragments into a vector at Once


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#1 The Gene

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Posted 01 June 2010 - 10:15 AM

HI GUYS I AM TRYYING TO LIGATE 2 FRAGMENTS AN ENHANCER AND A PROMOTER TO A VECTOR. THEY HAVE BEEN PCR AMPLIFIED WITH A RESTRICTION SITE TAG SUCH AS :


ENHANCER : EcoRIxxxxxxxxxxxxxxxxxxxxNcoI (digested)
Promoter : NcoIxxxxxxxxxxxxxxxxxxxxxBamHI ( digested)
Vector : Cut with Eco and Bam



Now i want to ligate enhancer along with promoter to a vector with sticky ends obviouslyat once
. can you people give you strategies or views that might help me in ligation??


Thank You
The Gene

#2 bob1

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Posted 01 June 2010 - 05:14 PM

ligate the two fragments first (how you will get 2 NCO1 ends binding I don't know, unless you polish), then ligate into vector.

#3 The Gene

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Posted 02 June 2010 - 10:42 PM

ligate the two fragments first (how you will get 2 NCO1 ends binding I don't know, unless you polish), then ligate into vector.



Hi thnx for replying. Wel im gona cut both enhancer and promoter separateyy with NcoI and gel elute them . Now they wil be havin sticky ends compatible.


So u sayin i should go for first ligation of these 2 fragments and then same mixture i should use for ligation with the vector ?

#4 fysio lab

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Posted 03 June 2010 - 12:52 AM

ligate the two fragments first (how you will get 2 NCO1 ends binding I don't know, unless you polish), then ligate into vector.



Hi thnx for replying. Wel im gona cut both enhancer and promoter separateyy with NcoI and gel elute them . Now they wil be havin sticky ends compatible.


So u sayin i should go for first ligation of these 2 fragments and then same mixture i should use for ligation with the vector ?


If you want to give it a try to ligate 3 fragments at once: add PEG8000 to the ligationmix ...you can try a 3point-ligation if your sites are not compatible

#5 The Gene

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Posted 03 June 2010 - 02:46 AM

ligate the two fragments first (how you will get 2 NCO1 ends binding I don't know, unless you polish), then ligate into vector.



Hi thnx for replying. Wel im gona cut both enhancer and promoter separateyy with NcoI and gel elute them . Now they wil be havin sticky ends compatible.


So u sayin i should go for first ligation of these 2 fragments and then same mixture i should use for ligation with the vector ?


If you want to give it a try to ligate 3 fragments at once: add PEG8000 to the ligationmix ...you can try a 3point-ligation if your sites are not compatible



My senior long time ago tried using 3:1:1 ( vector:insert1:insert2) mixture for ligation and it worked..what u guys suggest?




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