Sephadex G-50 column protocol
(to clean up cycle-sequencing reactions)

Preparation of Sephadex G-50 Fine Grade Slurry (Sigma S-5897- ABI Protocol):

1. Weigh out 10g of G-50 powder and transfer to a 250ml Erlenmeyer flask.

2. Add 150ml water and plug with a Kimwipe.  Mark the level on the flask to monitor possible volume loss during heating.

3. Microwave for 15 minutes, low heat (setting 4 on the Core Lab microwave).

4. Add a stirbar to the flask after microwaving and store in the refrigerator (4°C).

Discard the unused G-50 slurry after 3-4 weeks.  Old G-50 cracks more often and becomes concentrated because of evaporation.

Cycle-sequencing reaction clean-up

We re-use Centri-Sep columns and collection tubes from Princeton Separations.  These originally came with a commercial G-50 package and were intended for one-time use.  We repack them with the G-50 slurry prepared in the lab (see above).

1. Place the Erlenmeyer flask with cold (4°C) G-50 solution on a stir plate to keep the solution
from settling.  This is important when pipetting the G-50 into tubes in step 3.

2. Make sure Centri-Sep collection tube and column is clean and dry.

3. Pipet 650ul of the G-50 slurry (stirring on stir plate) into a Centri-Sep column.  Avoid bubbles by adding from the bottom of the column.

4. Spin column/collection tubes for 3 minutes at 2500 rpm.

5. Remove the column from the collection tube and place in a prelabeled (with your numbers) 1.5ml Eppi tube.  Write clearly on the tubes, the Core Lab technician must be able to read your scribble.

6. Add your cycle-sequencing product to CENTER of G-50 resin (not the side).  Do not touch the G-50 resin with the pipet tip.

7. Spin as before for 3 minutes at 2500 rpm.  You should see about the same volume in the bottom of the Eppi tube as was originally loaded on the column.

8. Dry the product on the Speed Vac with NO HEAT.

9. Shake out used G-50 and rinse column with deionized water.  Do not use hot water or detergents.  Soaking in DI water is OK.   Do not leave the tubes soaking in water overnight for someone else to clean up.

10. Samples cab be stored in a refrigerator or freezer until given to the Core Lab

The Core Lab resuspends your product in a 4.0ul dye/formamide mix.  The resuspended DNA is denatured at 90°C for 2 minutes and put on ice just prior to being loaded on a gel.