You can use the commercially available columns at your lab. Refer to the manufacturer's instructions for the procedure. The centri-Sep spin are available from Princeton Separations (P/N CS-901)

Spin Column Protocol

Column Preparation:

1. Pierce the bottom of a 0.5 ml eppendorf tube with an 18 gauge needle. Do not run the entire shaft of the needle through the tube; this will create too large of an opening.

2. Place the 0.5 ml tube into a 1.5 ml tube. (Repeat steps 1 & 2 for all reactions.) Prepare one or two extra columns in case some columns crack.

3. Add a 2-3 mm layer of glass beads to the 0.5 ml tube.

4. Add about 500 ul of Sephadex G-50 to the 0.5 ml tube.

5. Place the 2-tube assembly, caps open, into the Baxter microcentrifuge. Maintain a consistent orientation of cap lids through all spins (You can tuck in the 0.5 ml lid into an adjacent rotor well.)

Spin for 5 minutes at 2000 rpm.

6. Discard the elutant from the 1.5 ml tubes. Spin for 5 minutes at 2000 rpm.

7. Discard the elutant. At this stage the Sephadex (column) will have ‘pulled’ away from the sides of the tube. Columns with center cracks cannot be used.

Purification:

8. Label new 1.5 ml tubes. Discard the old 1.5 ml tubes. Place spin columns into the 1.5 ml tubes.

9. Add cycle sequencing extension product to the top center of the column. Do not touch the column with the pipet tip.

10. Spin for 4 minutes at 2000 rpm.

11. Discard columns. The 1.5 ml tubes should have approximately 10 ul of elutant. There may be more elutant if the column was still wet at step 8. More elutant will increase drying time but will not affect the sequencing product.

12. Place the 1.5 ml tubes, lids open, in the speed vac. Heat setting should be on medium. Close lid and press on. Sign in on the steno pad on top of the speed vac. Drying time is approximately 20 minutes. To check sample progress press off and wait for rotor to stop before opening speed vac lid. Tubes will be dry and you will not see a pellet.. (optional)

13. Add 12ul of deionized formamide to each sample. Aliquots of Hi-Di formamide labeled as "HD-F" are available from the top door shelf in the -20 C service freezer. Resuspend thoroughly by pipetting up and down. Vortex briefly. Spin down for 5-10 seconds.

14. Samples should have your initials and numbers. Please - no 10 digit clone names!

15. Place resuspended samples in the labeled " Capillary Sequencer-tube" box for individual tube users in the -20C service freezer.

16. Fill out the "Automated Sequencing Service Charge Form" on the desk in the sequencing area. Please fill out all the white blanks.

Samples not documented on the Charge Form will not be loaded.

17. An LPGT Technician will denature and load the samples. The capillary run times can vary depending on the number of samples.

* See the lab personnel for more information to set up the column method at your lab.

Extension Product Purification for 96-well plates Users

Isopropanol Protocol

You will need the following reagents and equipment for this procedure:

Variable speed tabletop centrifuge with microtiter plate, capable of reaching at least 1400 g.
Adhesive-backed aluminium foil tape (3M Scotch Tape 431 or 439)
75% Isopropanol (2-propanol) or 100% isopropanol (anhydrous) at room temperature.

Performing isopropanol precipitation:

1.Remove the 96-well reaction plate from the thermal cycler. Remove the Caps or pad

2. Add one of the following:

80µl of 75% isopropanol, or 20µl of deionized water and 60 µl og 100% isopropanol.
The final isopropanol concentration should be 60± 5%.

3. Seal the plate wiht strip caps or by applying a piece of 3M Scotch Tape 431 or 439 adhesive-backed aluminum foil tape. Press the foil onto the plate to prevent any leakage.

4. Invert the plate a few times to mix, or vortex.

5. Leave the plate at room temperature for 15 minutes to precipitate the extension products.

Note: Precipitation times < 15 minutes will result in the loss of very short extension products. Precipitation timnes > 24 hours will increase the precipitation of unincorporated dye terminators.

6. Place the plate in a tabletop centrifuge with plate adapter and spin it at the maximum speed, which must be 1400g but < 3000g.

1400-2000g : 45 minutes

2000-3000g : 30 minutes

IMPORTANT: Proceed to the next step immediately. If not possible, then spin the plate for an additional 2 minutes immediately before performing the next step.

7. Without disturbing the precipitates, remove the adhesive tape and discard the supernatant by inverting the plate onto a paper towel.

8. (optional) Rinse the pellet by adding 150µl of 70% isopropanol to each well. Seal the plate with adhesive tape and invert the plate a few times to mix. Place the plate in a tabletop centrifuge and spin at 2000g for 10 minutes. Remove the adhesive tape. Discard the wash onto a paper towel that is folded to the size of the plate.

9. Place the inverted plate with the towel in to the tabletop centrifuge. Spin until reaching 200g and stop the spin.

10. (optional) Add 12 UL of deionized formanide to each sample. Resuspend thoroughly by pipetting up and down. Vortex briefly. Spin down for 5-10 seconds.

11. Samples should have your initials and numbers. Please- no 10 digit clone names!

12. Place the plate containing vaccum-dried or formanide resuspended samples in the labeled "Capillary Sequencer-96 well plate" for large-scale 96 well plate users in the -20°C service freezer.

13. Fill out the "Automated Sequencing Service Change Form" on the desk in the sequencing area. Please fill out all the white blanks.

Samples not documented on the Charge Form will not be loaded.

14. An LPGT technician will denature and load the samples. The capillary run times for a plate will take about 5 hours.

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