Reagents

 

Binding buffer

7 M Guanidine-HCl in 200 mM MES buffer pH5.6 or
5.3 M Guanidine-HCl in 150 mM KAc buffer pH4.8

Glass fiber filter plate

Millipore multiscreen-FB filter plates, MAFB NOB 50

Catch plate

VWR, 622409-108

Wash solutions

80% Ethanol

Elution buffer

10 mM Tris pH 8.0

 

 

Protocol

  1. Aliquot 150 µl of binding buffer to 50 µl of PCR product. Thoroughly mix by vigorously pipetting up and down at least 5 times (complete mixing is important).

  2. Transfer mixture to 96-well filter plate.

  3. Put filter plate with centrifuge alignment frame (Millipore) on top of 96-well catch plate.

  4. Spin liquid through filter, into 96-well catch plate (1000x g for 5 min).

  5. Discard filtrate, save catch plate for reuse.

  6. Add 200 µl of 80% ethanol to each well of filter plate.

  7. Spin liquid through filter, into 96-well catch plate (1000x g for 3 min).

  8. Discard filtrate, save catch plate for reuse.

  9. Repeat 80% ethanol wash (steps 6-8) 1 to 4 times.

  10. Do one dry spin to remove residual ethanol (1000x g for 5 min).

  11. Add 50 µl of 10 mM Tris-HCl pH 8.0 (preheated to 65°C) to each well. Incubate 1 minute.

  12. To elute purified DNA, place the filter plate on top of a clean 96-well catch plate along with a centrifuge alignment frame and spin (1000x g for 5 min)

The PCR product is in the eluent. Transfer 10 µl to a clean catch plate with 10 µl DMSO in each well. Seal plates carefully with aluminium foil. Store both plates at -20°C.

Department of Microbiology
School of Medicine
University of Washington
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