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Oligo Synthesis >
Purification
Purification
As noted
here, every effort is made to maximize quality and yield in our unpurified oligos, and they are suitable for most PCR and sequencing applications without further
purification. When higher purity is required, two types of contaminants can be removed:
- organic salts (byproducts of blocking groups which protect the bases and backbone
during synthesis and are removed after synthesis is complete)
- failure sequences (small amounts of shorter DNA molecules lacking various
portions of the 5' end of the oligo) Failure sequences arise because the efficiency of
coupling is about 99% rather than 100% at each residue, and because the synthesis cycle
"caps" these failures to prevent their further extension. After deprotection
these molecules are normal DNA with hydroxyls at both ends.
Salts are readily removed by the researcher via simple procedures such as isobutanol
extraction, alcohol precipitation (not recommended for short oligos), gel filtration, or
passing the oligo through a reverse-phase cartridge (such as Sep-Pak), the latter being
probably the most effective method. We can provide a protocol for
Sep-Pak treatment.
Failure sequences can be removed by HPLC (for shorter oligos) or gel electrophoresis. A
third method, "OPC" cartridge purification, is partially effective in removing
failure sequences and simultaneously desalts the oligo. For this procedure, the trityl
group at the 5' end of the molecule is left on at the completion of synthesis and is used
as a hydrophobic "handle" to fish out the completed chains, since the failure
sequences will lack a trityl group. We can provide a protocol for this
procedure. If you wish to use this latter method, YOU MUST SPECIFY THAT THE OLIGOS ARE
TO BE RUN "TRITYL ON". There is no charge for leaving the 5' trityl on.
Gel Purification:
Gel electrophoresis is the most effective way
to remove failure sequences from most oligos. We now offer gel purification of
DNA oligos to both Yale and non-Yale investigators at all synthesis scales. Surcharges for purification are listed in our
price schedule.
Purification typically adds several days to a week to turnaround time depending
on our workload (current status for each oligo can be monitored using the
Check
My Orders button).
When completed, purified orders for Yale labs
will be delivered by courier to your P.I.'s delivery location.
Deliveries will be made once a day in the mid-afternoon. If no delivery location
for your lab has been specified, we will phone you when your order is ready and
you can pick it up at 300 George St. (room 2127). Non-Yale orders are shipped
via Federal Express Priority 1. In all cases, the current status for each oligo
can be monitored using the
Check My Orders.
If you prefer to gel purify the oligos yourself, a protocol is posted on our
Oligo Protocols
page. Oligos >100 bases will contain significant amounts of organic salts that
may cause smearing and/or poor band resolution. These oligos should be desalted
before attempting gel purification.
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