cDNA kit recommendations?
Michael L. Sullivan
mlsulliv at facstaff.wisc.edu
Tue Mar 27 11:10:37 EST 2001
>Please share your spin size fractionation tips with us. The Epicentre kit
>I used wanted me to do low temp agarose gels, which after many tries I just
>gave up on. Anything would be better than that.
As I mentioned in my previous post, rather than use Stratagene's sizing
column (essentially a pipette) proceedure to size fractioinate the cDNA, I
used Pharmacia's SizeSep400 spin column. One thing that was odd however,
was that the instructions for setting up the column to spin were, at least
in my hands, physically impossible (this was a year ago, so maybe they've
modified them by now). Their proceedure called for placing the column in a
15 ml corex tube and prespinning the column. For the size fractionation,
this was to be repeated except a microfuge tube was to be placed in the
bottom of the corex tube. However, the column simply would not fit in a
Corex tube and then fit in a centrifuge. What I did instead was to take a
disposable 15 ml polypropylene tube and cut it off with a razor blade at
the 10 ml mark. The spin column fit nicely in this, and then I put the
whole thing in a 50 ml conical tube. I used this for the prespin. Then I
just used a fresh cut-off 15 ml tube for the fractionation, collecting the
cDNA directly in the polypropylene tube (again, I place the whole assembly
in a 50 ml tube). The whole thing took less than half an hour.
Of the 100 ul of cDNA reaction I had applied, I recovered approximately 70
ul. As expected, the bulk of radioactivity was retained by the column. I
ran about 5% of the recovered material on an agarose gel and did
autoradiography-- the cDNA looked good (virtually all of it was between 500
and 4000 bp). I phenol/chloroform extracted the remaining cDNA and
precipitated overnight. I quantified the recoverd product on ethidium
containing agarose. I ended up with approximately 1.5 ug of cDNA-- more
than enough for the subsequent ligations.
Hope this helps. Let me know if you have any more questions.
Michael L. Sullivan, Ph.D
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706
(608) 264-5144 Phone
(608) 264-5147 Fax
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