Emulsion Breaking Using Centrifugation

(For one full run with as many as eight projects)

Roe Lab, University of Oklahoma

Version 5     Date: 06/19/2006

 

1.      With 12 channel pipette, add ~100µl isopropanol to the first 16 emulsion reaction wells.    Note:  Contents of 16 reaction wells correspond to a project.

2.      Use a syringe to transfer the emulsion-isopropanol mix from the first 16 wells into a 50ml corning tube.

3.      Refill the first 16 wells with ~200µl isopropanol to wash the wells.

4.      Transfer the mix into the same Corning tube (Cap Type: Flat Top; Corning Costar No. 430921, Fisher Sci. Cat. No. 05-538-67).

5.      Add isopropanol into the Corning tube to volume ~ 30ml.

6.      Repeat steps 1- 5 for the remaining seven (the second to the eighth) projects.

7.      Mix the contents in Corning tubes by hand.

6.      Centrifuge the Corning tubes at 3200rpm (2700xg) and room temperature for 4 minutes in the Beckman GS6R (or Beckman Allegra 6R) table top centrifuge.

7.      Carefully decant supernatant without disturbing the white pellet.

8.      Perform another ~30ml isopropanol wash for all the eight tubes.

9.      Centrifuge as above (step 6) and decant supernatant.  Repeat one more time, doing a total of three isopropanol washes.

10.    Add ~10ml 1X Bead Wash Buffer (BWB) into each of the eight tubes.

11.    Mix by inverting.

12.    Centrifuge the Corning tubes again at 3200rpm and room temperature for 4 minutes.

13.    Carefully decant the supernatant without disturbing the white pellet.

14.    Perform a second ~10ml 1X BWB wash.

15.    Centrifuge as before (as in step 12) and decant supernatant. We don't necessaryly see a need to repeat one more time, as doing a total of two BWM washes seem sufficient.

16.    Transfer the contents of Corning tubes into Oak Ridge tubes (Nalgene Oak Ridge Polypropylene Copolymer Centrifuge Tubes, 29 x 107mm, Actual brim capacity, 43mL, Nalgene No.: 3119-0050, Fisher Sci. No. 05-529-1D.

17.    Rinse corning tubes with 10ml 1X Enhancing Fluid and add to corresponding Oak Ridge tubes.

18.    Centrifuge contents in Oak Ridge tubes at 10,000 rpm for 5 minutes.   Note: Set the centrifuge’s temperature to room temperature.

19.    Carefully decant the supernatant into another eight Oak Ridge tubes.   Centrifuge at 10,000 rpm for 5 minutes.  Decant the supernatant.

20.    Divide contents in each Oak Ridge tube into two 1.5ml tubes. 

21.    Centrifuge at 10,000 rpm for 10 seconds.  Turn 180º, and centrifuge another 10 seconds.

22.    Remove supernatant. 

23.    Repeat step 20-22 until all beads are collected.

24.    Combine all DNA beads into eight 1.5 ml tubes.  (One tube for each project)

25.    Centrifuge contents of 1.5ml tubes.  Remove all but 100µl of 1X Enhancing fluid from each 1.5ml tube.

26.    Continue with 454 protocol steps.