Ligate with 01 enzyme site - (Sep/18/2007 )
Hi alls,
In my case, in mus be insert 1 fragment about 1 kb in the pCAMBIA vector (~17kb), I used Hind III to cutting vector and release my fragment from another vector. Vectors I treatment with CIAP (to remove phosphase group to supress vector self ligation), then normal ligate. Normally I will get 2 result, insert fragment will be insert 2 direction 5'-3' and 3'-5', I must be choose 5'-3' direction, but alls colony I get just only 3'=5' direction, I repeat my expriment 9 times but i can't choose any clon have 5'-3' direction. I don't know why, so can anybody explain for me about this case? Thank alls
well unfortunately, cutting by 1 enzyme drives no selectivity against the direction of cloning.
So 9 times are very boring i know, so i would suggest to create in that point a mini "multi-cloning-site". Add 4-5 restriction enzymes sites that don't match in your vector and/or in your insert. in theory,1 added site is sufficent.
so order 2oligos which should following those statements :
-complementary, they will have a hind III site and other(s)
-once oligos annealed, the borders should kill original hind III sites.
your oligos may not be ordered phopsphorylated (cheaper).
so after anneal, just phosphorylate them with T4PolyNucl Kinase their ready for ligation.
the procedure then will need to sewuence to know how is the orientation sense of these oligos, and if you have concatemerized. i did this before, and concatermer occured once. They are removable by double appropriate digestion and ligation without insert easily.
then you'll have 3 sites for directionnal cloning of your sequence. you will need to pcr your insert that time.
So 9 times are very boring i know, so i would suggest to create in that point a mini "multi-cloning-site". Add 4-5 restriction enzymes sites that don't match in your vector and/or in your insert. in theory,1 added site is sufficent.
so order 2oligos which should following those statements :
-complementary, they will have a hind III site and other(s)
-once oligos annealed, the borders should kill original hind III sites.
your oligos may not be ordered phopsphorylated (cheaper).
so after anneal, just phosphorylate them with T4PolyNucl Kinase their ready for ligation.
the procedure then will need to sewuence to know how is the orientation sense of these oligos, and if you have concatemerized. i did this before, and concatermer occured once. They are removable by double appropriate digestion and ligation without insert easily.
then you'll have 3 sites for directionnal cloning of your sequence. you will need to pcr your insert that time.
first I want to say thanks to mod, so in my case can not apply your way, I wil try 1 more time in my experiment, and just hope in lucky
In such situations there is no hope of luck. You will have to push things so that probability is on your side. How many colonies are you looking at in each experiment? I would look at maybe 72 colonies, or by colony PCR abou 96 clonies.
Unless you have exhausted all your colonies on the previous 9 plates, there is little reason to do repeat the experiment a 10th time. Because sadly, the 10th experiment will not be majorly different from the previous 9.
i'm sorry for the fact it can't apply for you... 17kp, yes it's quite long, but i'm wondering why? you may also use blunt end restriction sites if needed...
anyway, the point is to test more and more clones 
i've done that and know how it's frustrating, but you need to press this. also i press my thumbs for you
I don’t know if there could be some secondary structure or what, but it has happened to me some times. Heat at 50-60C and cold at room temperature before adding ligase has worked to me.
Good luck!
P.S.: Be sure you are characterizing properly.