Precipitation Protocols

2 No salt protocols for ABI BigDye:

Isopropanol:

1. Pipet the entire contents of each extension reaction into a 1.5 ml microcentrifuge tube. Volume must be 20 ul.

2. Add one of the following: 80 ul 75% isopropanol or 20 ul H₂O and 60 ul 100% isopropanol. Final isopropanol concentration should be 60 ± 5%.

3. Close tubes and vortex briefly.

4. Precipitate at room temperature for 15 minutes. Times < 15' may result in the loss of shorter extension products, times 24 hrs or at colder temperatures will increase the precipitation of unincorporated nucleotides.

5. Spin the tubes in a microfuge for 20 minutes at maximum speed. A pellet will probably not be visible, so care must be taken in subsequent steps not to lose the sample.

6. Carefully aspirate the supernatant. If doing more than several samples, an additional 2' spin before aspiration may aid in keeping the pellet down.

7. Add 250 ul 75% isopropanol to the tubes and vortex briefly.

8. Spin 5' max speed.

9. Aspirate the supernatant, then air dry or Speedvac remainder to dryness. Don't bring slightly wet samples over here without indicating it on the sheet, otherwise your sample may get diluted when we add loading buffer.

Ethanol:

1. As above

2. Add 16 ul H₂O then 64 ul 95% EtOH. Final EtOH concentration should be 60 ± 3%.

3.4. 5.6. As above

7. Add 250 ul 75% ethanol to the tubes and vortex briefly.

8. 9. 10. As above

These protocols are the recommended precipitation protocols from Applied Biosystems. They are inexpensive and do a good job at getting rid of dye blobs. However, note that care must be taken to avoid losing sample and multiple samples are sometimes hard to do.

DYEnamic ET Precipitation Protocols:

Note: we don’t do this, it’s right out of the manual. If you have questions call Amersham Biosciences tech support.

1. To 20 ul sequence reaction, add 2 ul NaOAC/EDTA solution (1.5 M NaOAC pH >8 [this is right out of the manual, we don’t know why it’s inexact], 250 mM EDTA)

2. Add 80 ul 95% EtOH and mix well

3. Centrifuge at rm temp 15’ at approx. 12,000 X g

4. Aspirate supernatant

5. Wash DNA pellets with 70%

6. Remove sup by aspiration

7. Air or vacuum dry 3-5’. Do not overdry.

Sephadex G-75

Note: Column cleanup with homemade columns can be an extremely economical way to process samples following cycling. We used to do this routinely in our facility, but have discontinued this in favor of magnetic beads which work better. We recommend the use of magnetic beads, but many users still run the columns with satisfactory results. Also, many companies (Amersham Biosciences, Qiagen, Princeton Separations, etc.) make commercial versions of these columns so if you choose this method you can investigate options from numerous vendors).

Materials:

1. Sephadex G-75 (fine; available through many distributors including Pharmacia and Sigma. G-50 Med or Fine also works)

2. Columns (96-well or used G-50 columns).

3. Autoclave

4. Centrifuge

Method:

1. Determine the number of columns that will be used.

2. Weigh out the sephadex and put it in an autoclave safe glass bottle.

(for approx 180 columns -> use 5g. Sephadex with 200 ml. of water)

3. Autoclave the mixture for 15 minutes and store in a refrigerator.

Note: Autoclaving will speed up the hydration of the sephadex and kill any fungus that may show up if the slurry is not used immediately.

4. Decant excess water after autoclaving so that there is approx. 1/3 water with 2/3 sephadex.

5. Transfer well shaken slurry to the top of each of the columns.

Note: Make sure the slurry is thoroughly mixed and uniform before adding it to a column. If this is not done, the sephadex “pillars” may not be uniform and the results from the sample clean-up may not be good.

6. Spin down the G-75 column at 2000xg for 1 minute to remove the water (if using a standard microfuge with variable speeds a setting of 5 out of 14 works well).

- Spin the 96-well plates at 750xg for approximately 2 minutes.

Note: The columns should be spun in a tube that it will collect the water from the hydrated sephadex. A waste tray should also be used for the 96-well tray.

7. Transfer the columns to either the sample collection tray (96-well) or a 1.5-ml labeled microfuge collection tubes.

8. Add the sample to the middle of the sephadex bed.

9. Spin the sample again at the same speed and time as in step 6.

9. Dry down the sample.

10. The remaining resin can be tapped out and the column re-used indefinitely. Further rinsing is not necessary. For the 96 well columns, it may be necessary to spray out some of the chambers to remove all the used matrix.

We have used the old Pharmacia AutoSeq G-50 columns as our columns—as far as we can tell they can be re-used indefinitely. Some manufacturers like BioRad and USA Scientific (Cat # 1415-0600 for USA Scientific) do make empty columns that can theoretically be used for this application. However, anecdotal evidence suggests it would be worth checking these out in advance since we have had reports of matrix getting through the frits.