bas at nospam.nl
Mon Mar 6 03:41:07 EST 2000
Kathryn Coyne heeft geschreven in bericht <38C12D0F.6F702000 at udel.edu>...
>Thanks for the input, Gys. To clarify, I am using biotinylated primers.
>My problem is not in removing the primers prior to concatenation, the
>problem is in generating the 102bp ditags before NlaIII restriction.
>I've been trying to troubleshoot and have come up with the following:
>my RNA and cDNA are of good quantity and quality based on gel
>electrophoresis; the kinase and ligase reactions seem to be working
>properly based on the test I did with the linkers by themselves. The
>only parts of the process I haven't tested are the initial NlaIII
>reaction, the BsmFI restriction, and the Klenow fill-in reaction. The
>restrictions are easy enough to check out, what about the Klenow? I
>admit it's been in the -20 for about a year, but the date is still good.
>It is also an exo(minus). (I'm assuming that the excess linkers are not
>being "blunted" then, since they have 3' overhangs, and that their
>complementary ends will ligate much more efficiently than the blunt
>ended ditags?). Maybe I should try T4 DNA polymerase. I also read
>somewhere that I could just throw in some Pfu and incubate at 70C for a
>few minutes to blunt the ditags instead of using Klenow. Has anyone else
>tried this? Any other ideas?
Hi Kathryn, your story suggests that there might be something wrong with
either the NlaIII restriction (of your 'crude' cDNA) or BsmFI restriction.
When NlaIII does not work (properly), you simply do not generate the correct
overhangs of your 3' cDNA ends. ALWAYS store NlaIII at -70 degrees Celsius,
and always add BSA to the digestion reaction.
When BsmFI doesn't work, you will simply not generate (enough of) the 102 bp
ditags, since the 3' cDNA ends are unaffected and do therefore not
contribute to the formation of ditags. BsmFI works best at 65 degrees
Celsius, requires also BSA, and should do just fine at that temperature. The
80 bp fragments you find on your gel are simply ligated linkers, the ones
that did not take part in any reaction except the ligation. They amplify
perfectly. Be glad that you don't find any other background products on your
gel! It took us quite a while to figure out that the quality of our primers
and linkers was really poor (although they had been PAGE purified). We
discovered that we were also amplifying primer multimers, and really weird
primer-linker stuff from which it was clear (after sequencing, that is) that
the sequences of the oligos were not always correct, thank-you-very-much! We
now routinely order our oligos with the same company as described in the
protocol from JHU, and suddenly all of our backgroung has disappeared.
Bas (in the same SAGE team as Gys)
basjhj at baserv.uci.kun.nl
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