DEBLOCKING AND PURIFICATION OF OLIGONUCLEOTIDES *
Sep-Pak:- *
Alternative Gel Purification:- *
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Oligonucleotides
DEBLOCKING AND PURIFICATION OF OLIGONUCLEOTIDES
Oligonucleotide is supplied still attached to the column. It should be detritylated, a choice usually made before synthesis and performed on the synthesiser. Alternatively detritylation could be manually performed by treating the column-attached oligo for a ver limited time with the TCA or DCA solution used on the synthesiser.
1. Quickly place 1 ml NH4OH (good quality 29+% kept at 4oC and opened only briefly to avoid loss) in a 1 ml syringe. Attach to column and place another 1 ml syringe at the other end of the column. Pass NH4OH through 4X.
2. Leave for 45 minutes at room temperature.
3. Pass the NH4OH through another 4X.
4. Leave for 75 minutes at room tempwerature.
5. Collect NH4OH now containing oligo, in a well sealed tube (eppendorf screw cap). Place at 55-58 oC overnight to remove the protective groups.
6. Dry in Speed-vac with heating (usually 3 - 6 hours).
7. Dissolve in 100 ul water for an expected yield of 1-5 mg depending on size.
8. Measure the OD at 260 nm and calculate the concentration assuming 35 ug of single stranded oligonucleotide gives an OD of 1.00 at 260 nm.
9. 1/3 to 1/2 of prep. is then purified on Sep-Pak C18 or purified bt gel electrophoresis:-
Solutions:
100% acetonitrile
25mM NH4HCo3
5% acetonitrile, 2mM NH4HCo3
5% acetonitrile
30% acetonitrile
H2O
a) Wash sep-pak cartridge with 10ml acetonitrile
b) Wash with 10ml H2O
c) Slowly apply oligo. prep. diluted to 1-3ml in H2O and repass through column 2-3 times
d) Wash with 10ml 25mM NH4HCo3
e) For oligos of >10-12mers, wash again with 10ml 5% acetonitrile, 2mM NH4HCo3 and finally with 10ml 5% acetonitrile. For smaller oligos wash with 10ml of H2O only
f) Elute slowly with 1ml 30% acetonitrile and again with a second 1ml of the same solution into a second tube. (Most of the oligo will normally be retreived in the first 1ml).
g) Lyophilise and resuspend in TE at desired concentration. (recovery after 5% acetonitrile wash will be typically 30% of original O.D. The full length product is however very efficiently recovered)
Alternative Gel Purification:-
Prepare a 6 M urea polyacrylamide gel in 1X TBE.
15% >20 mer
20% <20 mer
Add about 1mg oligonucleotide in 30 ul water to 60 ul formamide, boil for 3 minutes and then quench on ice. Load directly into a gel pockets about 20 cm long, 1 cm deep and 0.5 mm thick. Run gel under denaturing conditions, i.e. hot gel, until oligonucleotide is about 1/3 - 1/2 way down the gel. Load dyes in a separate pocket to give an idea of approximate mobility.
Separate gel plates and place gel onto Saran wrap. Take gel into dark room and place onto TLC plate or old intensifying screen and UV shadow to visualise the oligonucleotide. Cut oligo from the gel and place gel slice into 4 ml of 0.5 M ammonium acetate. Incubate overnight at 37 oC.
Recover ammonium acetate and wash gel slice with 1 ml of the same solution. Pool the solutions and recover using Sep-Pak C18 cartridge as above (solution can be applied directly or after dilution with water). Alternatively, oligo can be recovered by reducing volume to 100 - 200 ul by numerous dry butanol extractions. Add 5 - 10 volumes of absolute ethanol and place at -70 oC for 30 min. After spin wash with 80% EtOH and then 100% EtOH. Resuspend final pellet into 200 - 500 ul water. Measure OD to obtain concentration.