MILLIPORE MAFNOB 96-WELL PCR CLEANUP

Mike Deyholos (13 Jan 00) ; based on manufacturer’s protocol.

1. Orient the PCR source plate and MAFNOB filter plate so that well A1 is in the upper left-hand corner.

2. Mark the upper left hand corner of each plate with your initials and label the filter plate with the code that is on the source plate.

3. Place the filter plate on a vacuum manifold.

4. Add an equal volume of binding buffer to the PCR product in each well (usually 50uL).

5. Pipette up and down several times to make sure the solutions are well mixed.

6. Transfer the mixutre to the corresponding well in the filter plate.

7. When all 96 wells are filled, apply vacuum until the solution has been drawn through each of the wells. The exact vacuum pressure is not critical, but 200mbar works well.

8. Wait 30sec., then gradually release the vacuum.

9. With the vacuum off, apply 200uL of 80% ethanol to each well (wash 1).

10. Draw the ethanol through the filters as in step 7, and wait 15 sec. after the last well has been emptied before releasing the vacuum.

11. With the vacuum off, apply 200uL of 80% ethanol to each well (wash 2).

12. Draw the ethanol through the filters as in step 7, and wait 15 sec. after the last well has been emptied before releasing the vacuum.

13. With the vacuum off, apply 200uL of 80% ethanol to each well (wash 3).

14. Draw the ethanol through the filters as in step 7, but this time apply maximum vacuum (approx. 400 mbar), and wait 5min. before gradually releasing the vacuum.

15. Remove the filter plate from the manifold and blot the bottom of the filter plate on a clean kim-wipe.

16. Rinse and dry the vacuum manifold resevoir and reassemble the apparatus as before, with the filter plate on top.

17. Apply maximum vacuum for an additional 5min.

18. Turn off the vacuum slowly, to minimize backwashing ethanol through the filters.

19. Place the filter plate on a centifuge adapter, on top of an empty 96-well plate. Spin, with a balance plate, at 1000g (2500rpm) in the Sorval, at room temperature, for 5min.

20. Transfer the filter plate and adapter to a new clean, V-bottom, 96 well plate. Check the orientation of the plates and mark each with your initials in the upper left-hand corner. Label the new plate with the same code that was on the source PCR plate.

21. Pipette 120uL of 0.1X TE into each well

22. Wait 1 min. after the last well is filled, then spin again at 1000g (2500rpm) in the Sorval, at room temperature, for 10min., to elute the DNA.

23. Seal the V-bottom plate, and freeze at –20’C. Discard the filter plate.

Binding Buffer (500 mL)

334.4 g Guanidine hydrocholride (Sigma G4505; 99% pure; DO NOT SUB.CHEAPER GRADES !)

17.6 g MES free acid (Sigma M8250)

2.17 g MES sodium salt (Sigma M3885)

400 mL nanopure water

Stir with moderate heating until liquid is perfectly clear. It may be necessary to add an additional 50ml of nanopure water. A cloudy mixutre with insoluble material means the guanidine was impure; this will greatly reduce DNA recovery.

Adjust pH to 5.6 with NaOH.

Complete volume to 500mL.