Reagents:

Whatman 23x23
Boiling waterbath >95 °C
Denaturing Solution 0.5M NaOH, 1.5M NaCl
Neutralizing Solution 1M Tris-Cl pH 7.4, 1.5M NaCl
PROPK Solution 50mM Tris-Cl pH8.5, 50mM EDTA, 100mM NaCl, 1% Na-lauroyl-sarcosine
Proteinase K 500mg/9ml, use 3ml per 600ml PROPK

                       

 

Protocol:

  1. Observe membrane and note any abnormalities about growth after spotting. Check pencil numbers and robot puncture marks.

  2. Handle membrane with 2 forceps holding it for diagonal corners.

  3. Place on Whatman pre-wetted in Denaturing Solution. Leave for 4 minutes.

  4. Place membrane on fresh Whatman pre-wetted in Denaturing Solution and transfer to glass plate sitting on empty pipette tip box in boiling waterbath. Leave for 4 minutes with covered lid.

  5. Place membrane on Whatman pre-wetted in Neutratlizing Solution. Leave for 4 minutes.

  6. Place membrane on dry Whatman. Leave for 1 minute.

  7. Submerge membrane under a 600ml pre-warmed (37 °C) PROPK Solution containing ~150mg/ml fresh Proteinase K. Do NOT shake. Leave in incubator for 30-50 minutes.

  8. Place membrane on dry Whatman.

  9. Cover with a NUNC. Leave overnight.

  10. UV cross-link.

  11. Store between dry Whatmans.

Department of Microbiology
School of Medicine
University of Washington
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