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Solutions
Gel Stocks
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 NH 4OAc
up to 100 ml Q
Formamide Dye
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
Procedure
• 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.
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Labeling oligonucleotide