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 NH4 OAc

19.2 g NH4 OAc

up to 100 ml Q

Formamide Dye

9 ml deionized formamide

500 m l 10X TBE

500 m l 0.2% bromophenyl blue and 0.2% xylene cyanol

Other Reagents Needed:

0.22 m M 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 m l 10% APS (Gibco #15523-012), 20 m l TEMED (Sigma #T8133).

• Dry down 100 m g oligonucleotide and resuspend in 50 m l 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 m M 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 m l TE and quantitate 5 ml.