two step digestion - (Jun/25/2009 )

can any one provie me the protocol for the two step dna ligation....
thank you

two step ligation or digestion? For either of them, I am unware of any two step protocol.

Do you mean digesting with two different restriction enzymes?

for sequential digestion:

Add 1 volume NaOAc 3M, pH 5.2 to the digest and 6 volumes ethanol, keep for 2 hrs at -20, spin, wash with 70 % ethanol, dry

You have precipitated yr singly digested DNA, now setup the second digestion.

Best,
TC

You'd need to recover the product of the first ligation (linear molecule of size vector + insert) from a gel, and then use this in a second dilute ligation to maximize intramolecule ligation.

HomeBrew on Jun 28 2009, 10:15 PM said:

Base on the original post of roshanbernard, he gave a link for an article about 2 step ligation. I read the abstract, they mentioned using the high molar concentration for the first step, then dilute the mixture to lowering the concentration because after the 2 ends of vector and insert are ligated, the remaining step is the self ligation of the hybrid plasmid, this step prefers low molar concentration. I can't find the detail, but i think the overall scheme is right.

roshanbernard on Jun 24 2009, 09:42 PM said:

you mean dna ligation?
kindly check this site http://ivaan.com/protocols/131.html or this site
http://de.answers.yahoo.com/question/index...08100419AAgQy5G
i believe this can help a lot in what you've searching for..

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