How to ligate ds-oligos into a vector? - (Aug/25/2004 )
I point you to an older thread in the archives.
The punchline to your answer is that circular plasmids that exist in the form of supercoiled DNA move faster than linear DNA on a gel.
Also recently i cloned my gene in a vector. to confirm it i done gene release with double digestion. I then run gel with only vector on one lane and the digestion on other lane. I found that the double digested vector DNA in second lane is slighely above the control vector in the gel. what may be the reason...
I have been cloning about 100 RNAi-Vectors with oligos of 60 - 70bp.
Try that easy and reliable way:
1. order two oligos(regular and reverse-complement) with full restriction site sequence plus 3-5 bp overhang i.e.:
2. anneal 10µM in water 1 min 95 °C and cool down on bench. The concentration should about 100 ng/µl.
3. digets 1 µg with appropriate restriction enzymes and purify by phenol/cloroform extraction.
4. precipitate DNA by salts + 100% ice cold ethanol 10 min -20 °C.
5 determine concentration and use for ligation in digested + dephosphorylated vector.
For more information you can also use our detailed nature protocol .
hope this help
Uhm........ why do you use phenol-cloroform instead of a gel extraction kit?
Interesting tips though, I havent been purifying my digestion and I also havent dephosphorylated my vector because I figured it was ok if the double digest was pure enough......
Oh well if my attempt this time doesnt bring forth any colonies I guess its back to phenol-chloroform and phosphorylation for me
I use phenol-chloroform extraction plus ethanol precipitation instaead of kits because in my hands the yield of the dna is about 80-90% and the procedure works always!
Uhm...all I would need is the mskat buffer to do it like that
I read your paper, do you think there would be a difference if I used promega enzymes? Do you have any experience with those??? NEB doesnt seem to be very popular in the lab but if im going to buy a CIP and a kinase for my insert and vector and I buy NEB then I couldnt use the same buffer after digestion for the cip reaction right?
Im so glad you posted on here, i was beginning to lose hope hehe
Also another thing,
I anneal complementary primers so that they already have the overhangs so i dont have to double digest anymore. So my ds oligos arent phosphorylated, you dont mention an insert phosphorylation step so I guess yours are.
Uhm.....do you think this might be a problem, I mean the fact that I anneal primers with overhangs already so I dont double digest?
I never anealed oligos with overhangs, but you must phosphorylate them. You can use promega enzymes and CIP should work in this buffer.
As you mentioned MSKAT buffer is my favorite salt buffer for ethanol precipitation. It works and works and works....
lol i guess ill try it out then! Do you use fresh buffer everytime? Or do you keep a big bottle of it somewhere? hehe
big bottle stored elsewhere.