4 replies to this topic

[The Gene]

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

[bob1]

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

[The Gene]

bob1, on Jun 2 2010, 07:44 AM, said:

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 ?

[fysio lab]

The Gene, on Jun 3 2010, 08:42 AM, said:

bob1, on Jun 2 2010, 07:44 AM, said:

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

[The Gene]

fysio lab, on Jun 3 2010, 03:22 PM, said:

The Gene, on Jun 3 2010, 08:42 AM, said:

bob1, on Jun 2 2010, 07:44 AM, said:

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?