PROCESSING OF HIGH DENSITY BACTERIAL COLONY FILTERS FOR HYBRIDIZATION

This method is to use for preparing colony filters from BAC/PAC clones. The filters generated are suitable for hybridization with radiolabeled or biotin-labeled probes.

• Lysis solution - 2X SSC, 5% SDS

 For 500 mL:   50 mL 20x SSC, 125 mL 20% SDS, dH2O to 500 mL
 Make up with sterile stock solutions and store at room temperature.

• Pro. K Buffer: 50 mM Tris (pH8.0), 50 mM EDTA, 100mM NaCl, 1% laurylsarcosine

 For 500 mL:  12.5 mL 2M Tris (pH8.0), 10 mL 5M NaCl, 5 g N-laurylsarcosine, 
 50 mL of 0.5 M EDTA, dH2O to 500 mL. Filter-sterilize and store at room 
 temperature.

• Bio-Assay Dishes: 1.5% Agar, 25 g Lura Broth in 1L ddH2O.

 For one 22 cm2 dish, pour 300 mL of 1.5% LB/Agar with appropriate 
 antibiotic added. Autoclave and let it cool to reasonable temperature 
 for adding antibiotic before pouring.  Run liquid 60cycle for a 2 L volume.

• Proteinase K (10mg/ml or 20 mg/ml stock) (store at -20o0)
• Microwave oven
• Nunc Bio-Assay dishes (#240835)
• Nylon membrane (Schleicher & Schuell, NTYRAN SPC, #10416270, or Genetix #N3010)
• Large square glass plates
• Large Tupperware dish with lid
• 3mm chromatography paper
• Stratalinker

Prepare plates one day before using them.

• Use the BioGrid to apply clones to 22 cm2 nylon membrane, which have been overlaid onto 300 mL of LB/Agar (with appropriate antibiotic) bio-assay dish. Grow the colony filter overnight at 37oC.

• Place a piece of 3 mm chromatography paper (slightly larger than nylon membrane) onto a large square glass plate (which is placed within a cafeteria dining tray .) Saturate the 3 mm paper with lysis solution (above). Flatten the 3 mm paper so there are no bubbles (can use a 10 or 25 mL pipet to do so.)

• Lay the colony filter, colony-side up, on the 3 mm paper. Allow to sit for 3 minutes at room temperature. The colonies will begin to appear very mucoid

• Place the entire filter set onto a turntable in a microwave oven and microwave at maximum power for 2-21/2 minutes . If there is no turntable in the microwave oven, the baking dish should be rotated 120o every one minute. The colonies should appear flattened on the colony filter and the edges of the filter should be curled up. If the filter is still damp, continue microwaving until completely dry.

• This step requires 100-200 ml of proteinase K solution per four colony filters. In a large Tupperware or Pyrex baking dish, add the required amount of proteinase K buffer (above). Add proteinase K to a final concentration of 10 µg/ml (for 100 ml of proteinase K solution you will need 100 µl of a 10 mg/ml stock or 50 µl of a 20 mg/ml stock). Add each filter individually, swirl the dish so that each filter is completely covered. Cover the container with a lid or with saran wrap. Incubate at 37oC for at least two hours, rocking the dish occasionally. After this incubation, colonies should appear as indentations in the filter and there should be no bacterial debris. If the colony debris still seems to be stuck to the filter you can continue to incubate (up to 24 hours, if necessary).

• Rinse the filter in a large volume of 2x SSC by dipping in the solution briefly. Standard is to perform UV crosslink at this point then air dry on a used chromatography paper. Dry each filter without overlying each other: filters would be badly stuck and cannot be separated when they dry one on top of another. There is no need to UV crosslink with this method; however, if the filter is probed several times, UV crosslinking should be performed using the “autocrosslink” setting on the Stratalinker while the filter is still damp.

You could use any large tray/dish for this purpose as it merely serves as a sink for excess lysis buffer. Note: it is a good idea to check the membrane after 2 minutes to make sure it is not completely dry. If it is, remove the membrane from the microwave and proceed to step 5. If the membrane is still damp, continue with the microwave step. Adjust power of the microwave oven, if necessary, so that you won’t burn the filter at all.