Diluent 5X Buffer 25% Acrylamide
209 g Urea 209 g Urea 209 g Urea
up to 500 ml Q 250 ml 10X TBE 120.8 g Acrylamide
up to 500 ml Q 4.1 g BIS
up to 500 ml Q
2.5 M NH4OAc
19.2 g NH4OAc
up to 100 ml Q
9 ml deionized formamide
500 ml 10X TBE
500 ml 0.2% bromophenyl blue and 0.2% xylene cyanol
Other Reagents Needed:
0.22 mM disposable syringe filters, short wave UV source, intensifying screen for UV shadowing
Pour a 20% denaturing gel: 12 ml 5X Buffer, 48 ml 25% acrylamide, 500 ml 10% APS (Gibco #15523-012), 20 ml TEMED (Sigma #T8133).
Dry down 100 mg oligonucleotide and resuspend in 50 ml Formamide Dye.
Boil for 2 minutes and hold on ice until ready to load gel.
Run the gel at 15 W constant power until the fast dye is at the bottom of the gel.
To visualize the bands, UV shadow on the intensifying screen. Excise the band and crush and soak in 1 ml 2.5 M Ammonium Acetate at 37°C for several hours to overnight.
Spin for 10 minutes and remove the supernatant. Add another 1 ml of 2.5 M ammonium acetate, pellet and pool with the previous eluate.
Filter the supernatant through a 0.22 mM filter, add 0.1 volumes of 3M NaOAc 5.2 and 2 volumes EtOH. I usually add 1 ml glycogen also.
Spin for 30 minutes, wash and dry. Resuspend in 50 ml TE and quantitate 5 ml.