Date: Thu, 09 Jan 1997 15:19:25 -0700 (MST)
From: "Terry Mulcahy 272-5792; fax 272-9107" <TMULCAHY@medusa.unm.edu>
Subject: DNA Synthesis
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Date: Fri, 18 Oct 1996 18:22:14 -0400
From: "\"\"Dr. Anthony Yeung\"\"" <yeung@deneb.rm.fccc.edu>
Subject: Re: Oligo deprotection
In-reply-to: "Your message of Fri, 18 Oct 96 11:14:08 -0800."
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Hi Stephenie:
We have found that 10 hours at 55 C is acceptable for almost all the
oligos we have made. We use fresh ammonia only. However, we routinely use 4
hours at 65 C. Most people will settle for half that long. Much depends on the
length of the oligo you are deprotecting. Adenosine and cytosine are base
labile. NaOH treatment of DNA is used as an A and C specific reaction in
chemical DNA sequencing. If your oligos are long, incomplete deprotection at
standard duration can become a problem that you have to trade off with alkali
degradation. You need to ask the tough question: Am I or my user better at
purifying not fully deprotected oligos from the perfect oligo, or are we
better at separating alkali damaged oligos from the perfect oligos? You can
ask what the oligos will be used for. For example, incompletely deprotected
oligos will be competitative against the perfect oligo in a PCR reaction
whereas the alkali damaged oligo is transparent in that reaction. In cloning,
the incompletely deprotected oligo will be non-clonable most of the time while
the alkali damaged oligo may be more mutagenic. The problem theoreticall gets
worse as the square of the lengths of the oligo. Fortunately, most oligo
applications are robust and an oligo really has to be bad to be detrimental.
The two exceptions may be crystallography and when mutagenesis is attempted
with the mutation near the 3' end of the oligo. Yes, 24 hours is too long if
sometimes you have several C in a row.
Hope it helps.
Tony
>Regarding post-cleavage deprotection at 55 degrees, how long is too long? Can
>they stay at 55 for 24 or more hours without degrading? Thanks.
>
>Stephanie Willis
>Shriners Hospital Research Unit
>Portland, OR
>
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Anthony Yeung, Ph.D. Phone: (215) 728-2488
Member FAX: (215) 728-3647
Fox Chase Cancer Center Internet: AT_Yeung@fccc.edu
7701 Burholme Ave.
Philadelphia. PA 19111
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