Dr. Peter Gegenheimer
PGegen at kuhub.cc.ukans.edu
Fri Jul 18 17:56:07 EST 1997
In <lvracxt2jq.fsf at zifi.genetics.utah.edu>, zinc <zinc at zifi.genetics.utah.edu> writes:
>we've seen a little of this sort of thing in our lab. it seems to be
>an artifact of the transcription. do your RNAs start with three Gs? T7
>often stutters with those and the result is a non-homogeneous pool of
>RNAs that give 'shadow' bands.
>Matt Thomas <Thomas at molbio.uoregon.edu> writes:
>> Does anyone know of conditions where RNase T1 will cut other then after
>> a G? IT is advertised that it cuts only after G's but I have some
>> strange digestion patternes which suggest sometimes it cuts weird. The
>> DNA sequence that I make the RNA is fine, and I'm checking the RNA
>> directly with primer extension, but I want to rule out that it is the T1
>> that is doing strange things.
>> Matt Thomas
1) T7 RNA polymerase does NOT stutter at the GGG sequence in its promoter. (These are the last 3 bases of the promoter itself.) Stuttering usually requires 5 to 7 G's in a row. On the other hand, the appearance of "shadow" transcripts is caused by the non-template-directed addition of a random nucleotide to the 3' end of the transcript. Usually only one residue is added, but all 4 bases can be added. Not all templates show this!
2) RNase T1 is reasonably specific for Gp/N. You can see overdigestion with high concentrations of T1; typically cleavages will occur a few bases 5' of a G. To obtain clean digests, use 5 to 7.5 U of RNase T1 per 20 ug of RNA (the total [RNA] is brought up with carrier yeast RNA). DIgestion is in 10 mM Tris, 1 mM EDTA, pH 7.5-8.0 for 30 min at 37 to 50 degC.
-- You should also realize that too little RNase will result in partial digestion products, as well as a mixture of oligos ending in --G(3')p and the same oligo ending in --G(2',3'cyclic)p ("G>p").
| Dr. Peter Gegenheimer | Vox: 913-864-3939 FAX: 913-864-5321 |
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