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Double-stranded oligo ligation - (May/30/2007 )

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What I noticed, is that you do the annelaing different from me. I always use this protocol fo siRNA oligo annelaing and it has always worked fone for me.
Anneal the two oligos. Prepare a 20 μl annealing reaction in the following way:
1 μl top-strand oligo
1 μl bottom-strand oligo
1 μl 20 x SSC (Sigma, Cat. S6639)
17 μl water
5. Heat the mixture at 95 oC for 10 min. Take it out and leave it at room temperature for one hour. Dilute the mixture to a final insert concentration of 40 ng/μl with 30 μl of TE buffer .
_________________________
20 x SSC:

175.3 g NaCl
88.2 g sodium citrate

in 800 ml of H2O

Adjust the pH to 7.0 with a few drops of a 14 N solution of HCl. Adjust the volume to 1 liter with H2O.

Dispense into aliquots. Sterilize by autoclaving.

-Jou2007-

Friends, I have problems....because the oligos are already in my lab and I can not order it again.... unsure.gif

and I ordered the oligo without think in let it already ends cohesive....my oligo must to be cut for ligation.... dry.gif dry.gif

What should be the protocol more efficient having the dsoligo in these condition?
the enzymes for cut are HindIII and EcoRI?

thank in advanced

PLEASE HELP ME!!!


QUOTE (Jou2007 @ Jun 5 2007, 10:28 AM)
What I noticed, is that you do the annelaing different from me. I always use this protocol fo siRNA oligo annelaing and it has always worked fone for me.
Anneal the two oligos. Prepare a 20 μl annealing reaction in the following way:
1 μl top-strand oligo
1 μl bottom-strand oligo
1 μl 20 x SSC (Sigma, Cat. S6639)
17 μl water
5. Heat the mixture at 95 oC for 10 min. Take it out and leave it at room temperature for one hour. Dilute the mixture to a final insert concentration of 40 ng/μl with 30 μl of TE buffer .
_________________________
20 x SSC:

175.3 g NaCl
88.2 g sodium citrate

in 800 ml of H2O

Adjust the pH to 7.0 with a few drops of a 14 N solution of HCl. Adjust the volume to 1 liter with H2O.

Dispense into aliquots. Sterilize by autoclaving.

-javo-

the oligo annealing conditiions are the same as mentioned before.
You simply need to digest the annealed primers after that.

-perneseblue-

an efficient way to do this is to digest half of annealed by Hind III and the other half by EcoRI for 3hours (or O/N).
Then mix both preparations, and readd 0.5┬Ál of each enzyme.
Procedure is dons as that, because we guess that if enzymes are mixed together right from the start, there may be competition for enzymes to get the oligo as it may structure itself...

-fred_33-

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