(Dis) Advantages of 33-P
bl275 at cleveland.Freenet.Edu
Sat Mar 12 10:18:46 EST 1994
The other day I wrote:
>I find that me 33-P sequencing reactions go to pot after a day or two at
>-80 'C. I have used 33-P in PCR and other applications, and while its an
>easy isotope to work with, I have found it necessary to do any work with it
>quickly, before those funky bands rear their heads. With 35-S and 32-P,
I have been asked about these 'funky bands' in 33-P sequencing gels. While
fresh 33-P sequences look nearly as good as those done with 35-S, those
stored for a day or two can look pretty bad. Almost as bad as I look when
I get up in the morning - ugh!
Decomposition of 33-P to 33-S occurs at a level of a few percent a day.
Since most of us use labeled dATP(alpha 33-P), this means that where 33-P
decomposition to 33-S occurs, the phosphodiester is converted to a sulfonic
acid ester just before dA residues:
5'-dN~O-P-O~dA-3' -----> 5'-dN~O-S-O~dA-3'
The problem occurs when you heat the sequencing reaction at 75-80 'C in
preparation for loading. At this point the sulfonic esters are hydrolyzed,
with the result that sequence pattern is littered with bands across all
four lanes everywhere an 'A' occurs. Not a pretty sight, my friends,
enough to make a biochemist cry. The heat lability of sulfonic acid
esters may not interfere with its use in other molecular biological
applications, but in sequencing, 33-P has a definite disadvantage.
I plan to stick to [35-S]dATP(aS). If you will be running your reactions
the same day, 33-P may be fine. If you are trying to work out a difficult
stretch of sequence which may require formamide gels, 36 hour runs and the
like, it may be more useful to run your Sequenase reactions at four times
their normal volume with 35-S and keep them at -80 'C. They will be usable
for several weeks and you wont have to repeat the reactions when you decide
to try different electrophoretic conditions to get those 500 bases from the
primer (I am sure that there are other sequence hogs out there).
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