Frank O. Fackelmayer
Frank.Fackelmayer at uni-konstanz.de
Mon Feb 14 04:18:53 EST 2000
S Hopkirk wrote:
> Hi, just wondering if you can help me with a couple of questions about oligo
> cloning, as I too am trying to cloning using this method.
> My your method describes dissolving oligos to 100pmol/uL.
> My PCR product is ~2000bp and I estimate from gel electrophoresis of purified
> DNA that I have it in solution at a concentration of 6ng/uL. How do I convert
> this to 100pmol/uL?
> Also when you precipitate the cut vector in EtOH, do you follow the method in
> Manniatis, and if so do you usually get to see a pellet once you have done
> this? When I precipitated 1ug of cut vector, the pellet wasn't visible.
> Thank you,
As to the calculation: When you have a 2000bp product, the mw is 2000 * 660 (mw
of one base pair) = 1.320.000 g/mol. So if your sample has a concentration of
6ng/ul (or 6mg/l = 6e-3g/l), that is 6e-3/1.320.000= 4.5e-9M or 4.5nM. Thus,
there are 4.5nmoles in one liter, or 4.5fmoles in 1ul.
However, I don´t really understand your question. What I was talking about was
cloning of oligos, not PCR products, into a cut vector. In my protocol, I adviced
to use 1ug of cut vector. For a usual vector of 3kb, the calculation is:
MW= 3000*660= 1.980.000, so 1ug is 1e-6/1.980.000= 5e-13mol or 0.5pmol. The
protocol calls for 10ul of oligos at a concentration of 100pmol/ul (or 1000pmol
in total), there will be an oligo excess of 1000/0.5= 2000fold. This is enough to
essentially block all religation of the vector, and gives a high yield of correct
constructs (usually >80% in my hands).
As to the precipitation: Yes, I use a standard EtOH precipitation as outlined in
the major cookbooks (add 1/10 vol of 3M sodium acetate, pH 5.5, mix, then add
2.5vol of cold EtOH, let stand, centrifuge for 10min full speed (10000g), air-dry
pellet). If your DNA is clean enough, you will hardly see the pellet of 1ug.
Don´t be afraid, the invisible pellet sticks to the wall of the tube quite well,
so it is not a problem to remove the supernatant by aspiration.
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