cloning shRNA - help (Jul/30/2005 )

hi all
i am trying to clone shRNA insert (80 bp) in a 3kb plasmid. for this i need to cut it with BseR1 and BamH1.
the problem is that i am not getting clones at all. Both the sites are situated very close so i cant make out whether the plasmid is properly digested. i first digest it with BseR1(since it needs dummy sequence) and cut purify the plasmid using quiaquik.then i set up BamH1 digestion.
will anybody tell me how to exactly calculate the moar ratio of 1:3??
for example if i have 100ng/ul vector and 10 ng /ul insert, how much of vector and insert should i take??
also i am getting very less annealing of single stranded shRNA. how can it be improoved?? i cut purify the annealed oligos and then kinase them.then set up ligation
thanks

hi
first for your ligation is to define the amount of plasmid you use.
In most cases it's 100ng.
That says you need 1µl of your prep.
Assuming your plasmid is 3000pb and your insert is 80pb, your plasmid is 3000/80 times bigger than your insert.
Hence for 100ng of plasmid you'll need 3times more insert in molar unit.
If your insert and your plasmid have the same size you'll need 300ng of insert. But your insert is more little as your plasmid in proportion 3000:80.
You'll need so : 3x100ng / (3000/80) = 300*80/3000 = 8ng.
You'll need 0.8µl of your insert prep.

For your digestion : After the first digestion, i think you'd better do a precipitation of your plasmid and purify on gel the final product. I do so because i think a gel purification is not that clean and may interfers with a second digestion, especialy if your sites are close.

for annealing : i heat my preparation at 95°/5' in a heating block and let all stuff (prep + block) gently cool at room temperature (i maintain my eppy in the heating block causthe decrease is slower and enhance the annealing process.
but i would recommend you to buy pre phosphorylated oligos. More expensive but better for this stuff (and you are quite sure that all molecules are phosphorylated, a step you are not 100% sure in a manual phosphorylation...)

Hope that helps you to solve your pbs.
Fred

thanks
i have tried it. when i anneal the oligos as you have suggested, and kinase them, i run them on a 2% agarose gel. i cut the annealed speies and leave the single stranded species on the gel. using quiaquick colums, when i elute the DS oligos, and run on the gel for quantification for ligation, i again see two species of oligos. one runs at 80bps and another on 50 bp.
pleas esuggess as why it is happening??? i thought there is a problem in elution buffer. i used 10mM tris pH8.5 instead but the problem remains same.

hi
just before runiing smples, heat them at 65° 5'.
Ds oligos are self complimentary and can form secondary structures that affect mobility.
fred