s.hopkirk at virgin.net
Mon Feb 14 13:51:56 EST 2000
Thank you for replying in such a detailed way. Yes I am a novice at cloning so it is
very helpful when the method is described in full.
As for the oligo cloning I just thought I would try your method with a PCR product to
see if it would work. I thought as you are annealing the two oligo's together it
could be the same as having a double stranded PCR product?
Do you do PCR cloning as well ?
"Frank O. Fackelmayer" wrote:
> 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,
> > Sarah
> Hi Sarah,
> 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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