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Will Crude Oligos Work For
You?
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What are crude oligos?
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 Crude oligos contain
all the final products after completion of synthesis, cleaving, and
deprotection of the oligo. This would include the final correct length
product, all the n-1+mers, organic salts, and any residual synthesis
reagents.
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What are the n-1+mers?
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Phosphoramidite
chemistry is not 100% efficient. Oligo synthesis proceeds on a starting resin
whereby each base is added one at a time in the 3'-5' direction. Each time a
base is added, the coupling efficiency is not 100%. This efficiency depends on
the position of the base in the chain, the base itself, the length of
sequence, the individual machine, and how fresh the chemicals are on the
machine. Coupling efficiency is on average 98-99%.
Any growing chain
that fails coupling during an individual cycle is blocked from continuing to
grow by a capping step after each base addition. These truncated chains are
the subsequent n-1+mer species found in the final crude product. The following
is a chart that represents the percentage of full length oligo found in the
final crude product based on the length of the oligo and the average
efficiency during synthesis.
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Oligo
Length |
Eff |
| .995 |
.990 |
.985 |
.980 |
| 20-mer |
91% |
83% |
75% |
68% |
| 50-mer |
78% |
61% |
48% |
37% |
| 75-mer |
69% |
48% |
33% |
22% |
| 100-mer |
61% |
37% |
22% |
14% |
| Can I use Crude Oligos? |
| Standard
PCR and Sequencing: Crude oligos under 30 bases are
acceptable. If your PCR and Sequencing reactions involve large
templates such as genomic or BAC DNA, then we suggest OPC or PAGE
purified primers due to the increase in possibility of other potential
binding sites for the n-1+mers. |
| Cloning
and Site Directed Mutagenesis: Primers should be PAGE purified. |
| Labeled
DNA: Should always be purified. |
ABI 394, Crude, M13 Forward-21/pGEM
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ABI 394, OPC Purified, M13 Forward-21/pGEM
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Oligonucleotide Synthesis Service
Laura Guest - Senior Research Specialist
334 Edward R. Madigan Laboratory, 1201 W. Gregory Drive, Urbana, IL 61801
Phone: (217) 244-1007 FAX: (217) 265-5066
Email: gen-engr@uiuc.edu
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| Last edited:
29 Nov 2004
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