Section on Neural Gene Expression

l Phage cDNA Library Screening with ³²P-UTP Riboprobes

A. Bacterial cell maintenance

1. Streak out Y1090r- cells from glycerol stock onto LB agarose/ tetracycline (10µg/ml) plate. Store at 4°C in darkness for up to 2 weeks.
2. For bacterial cell use during library plating, grow up a single colony of Y109or^- cells in LB plus 0.2% maltose/10mM MgSO₄ until A₆₀₀ of 0.6 and 0.8 is reached (should take 3 to 4 hours). Store cell stock at 4°C (may be used for 2-3 days but better if used fresh).

B. Phage library titering<

Determine the l phage library titer by plating out dilutions of the phage lysate (assuming the library is at least 10⁸ pfu/ml). Serially dilute library 10^-6, 10^-7, 10^-8 and 10^-9 in SM buffer and incubate 100ml of each dilution with 200 ml of Y1090r- cells for 15 min at 37°C. Add phage lysate/cell mix to 3ml of molten 0.7% top agarose/10mM MgSO₄ (warmed to 55°C), invert quickly 3 times and pour onto prewarmed 10cm LB agar plates. Use duplicate plates for each dilution. Once the top agarose has set, invert the plates and incubate at 37°C overnight.

C. Primary library screening

1. For the primary library screen, aim to plate approximately 10⁶ pfu in total. If using 15 cm plates, dilute library to obtain 50,000pfu per plate (use 14 to 20 plates per screen).
2. For each plate, incubate 600µl of Y109or^- cells with 50,000pfu for 15min at 37°C. Add the lysate/cell mix to 10ml molten 0.7% top agarose/10mM MgSO₄ and plate out as described above. (For cDNA libraries, incubate plates inverted for approximately 7 hours; for genomic libraries, incubate for 10-12 hours.)
3. The next day, chill plates (at 4°C) for at least one hour before doing colony lifts. Make sure plates are labelled, and also label reenforced nitrocellulose filters (for duplicate lifts, label filters "a" and "b"). Saturate 2 layers of Whatman 3MM paper with denaturing solution in a dish (without having too much excess liquid), and repeat with two other dishes containing neutralizing and rinsing solutions, respectively. Carefully place filter "a" on cold top agarose of the plate and puncture with sterilized needle asymmetrically (3 places) so filters and plates can be oriented later. Let sit for one minute. Remove filter and place it plaque-side down onto the denaturing solution-saturated Whatman paper for one minute, and then for 3 minutes in the neutralizing and rinsing dishes, respectively. For "b" filters, allow them to sit on the top agarose for 2 minutes before proceeding. Replace the Whatman 3MM paper and solutions every 10 filters. Let air dry, place filters between clean pieces of Whatman 3MM paper, then dry in oven under vacuum for 2 hours at 80°C.
4. Prior to prehybridization, rinse the filters in 1xSSPE. Prehybridize filters overnight in 100-120ml of prehybridization solution, contained within 15cm crystalline dishes. Incubate at 65°C in a shaking waterbath. Repeat process with hybridization solution plus ³²P-UTP riboprobe (2x10³ dpm/ml) the following night.
5. Wash filters 4x30min in 0.1%SDS/0.1xSSC at 65°C, place on Whatman 3MM paper to blot excess liquid and wrap in Saran Wrap. Expose to film at -80°C for several hours and/or overnight.
6. Line up filters with plates and locate positive plaques. Cut out 0.5cm² agarose plugs around positives and place in 1.5ml SM buffer. Add 50µl CHCl₃, vortex and allow plaques to diffuse into SM buffer for 3-4 hours at room temperature before proceeding to second round plating. Store plugs at 4°C.

D. Second round screen

1. Grow up Y109or^- cells as before (see A.2.).
2. Dilute the the diffused phage lysate 10 ^-4 in SM buffer, and use 5ml and 20ml with 200ml of cells. Aim to obtain 200-500 plaques/plate. Plate onto 10cm plates as before (see B.1.) and incubate overnight at 37°C.
3. Repeat colony lifts using smaller filters. Prehybridize and probe filters in 30-50ml of prehybridization and hybridization solutions, respectively, using 10cm crystalline dishes. Check for, and isolate positive plaques. Start overnight culture of Y109or^- cells for final screen.

E. Third round screen

1. Plate final round so as to obtain 50-100 plaques/10cm plate (eg; dilute phage lysate supernatant 10-3 and plate 20ml and 5ml with 200ml of cells). Screen for positives and isolate single positive plaques with sterilised Pasteur pipette tip. Place single plaque-plug in 1.5 ml SM buffer with 25ml chloroform and store at 4°C.

SM buffer (for 400ml):

20ml 1M Tris, pH7.5 (50mM)
10ml 4M NaCl (100mM)
3.2ml 1M MgSO₄ (8mM)
0.04g gelatin (0.01%)

Denaturing solution: 0.5M NaOH/1.5M NaCl

Neutralizing solution: 1.5M NaCl/1M Tris-HCl pH7.4

Rinsing solution: 2x SSC

Prehybridization and hybridization solutions (for 40ml):
20ml 50% formamide
2ml 1X SSPE (20X)
8ml 10% Dextran Sulfate (50%)
4ml 5X Denhardt's (50x)
400µl 250µg/ml BRL tRNA (25mg/ml)
500µl 250µg/ml Sigma fraction XI total yeast RNA (20mg/ml)
400µl 100µg/ml single-stranded DNA (10mg/ml)
400µl 0.1% SDS (10%)
4.3ml water

Stock solutions are RNase-free (DEPC treated) and filtered sterilized where possible.

F. Lambda DNA Preparation

1. At end of day, take 200ml eluted plaque and add 200µl of Y109or^- overnight culture and 200µl Mg/Ca (10mM each MgCl₂ and CaCl₂). Incubate 15min at 37°C with mild shaking. Add 10ml LB/MgSO₄/maltose and incubate overnight with mild shaking at 37°C.
2. In the morning, add 1.25ml 4M NaCl. Mix gently, but completely. Add 25µl CHCl₃ and shake for 15min. Spin down debris at 4500rpm for 5min and save supernatant as plaque suspension for next step and for future use.
3. In a 250ml flask, place 40µl plaque suspension (may use phage suspension from step 15 of a previous prep to get faster lysis), 800µl Y109r- overnight culture and 800µl Mg/Ca. Incubate at 37°C with mild shaking for 15min (put horizontal in shaking incubator). Add 40ml prewarmed LB/MgSO₄/maltose and incubate at 37°C in shaker until lysis occurs (about 3 hours). Should see lumpiness and strands of fibers instead of swirly haze, or greater graniness. Lysis is usually 30 to 45min after first signs.
4. Add 5ml 4M NaCl, transfer to 50ml tube and add 50µl CHCl ₃. Shake gently at 37°C for 15min and spin 4500rpm for 5min.
5. Pour off supernantant into fresh 50ml tube. This is the phage suspension. Place 35ml into polycarbonate (Oak Ridge) tube with 3.5g PEG 6000. Dissolve by shaking on rotating mixer set on high for at least 5min. Incubate on ice for 60min.
6. Spin 6000rpm for 15min in swinging bucket rotor. Pour off supernatant and drain well. Resuspend in 400ml TM buffer (50mM Tris, pH 8, 10mM MgSO₄) by pipetting and vortexing. Transfer to microfuge tube, add 500µl CHCl₃ and vortex. Spin 10min and remove supernatant leaving thick interface.
7. Add 5µl RNase A (10mg/ml) and 2.55µl DNase (2.5U-RQI, Promega) and incubate 37°C for 60min.
8. Add 1/5 volume (~90ml) of phage lysis buffer (0.25M EDTA, 2.5% SDS, 0.5M Tris, pH 9), vortex, and heat at 65°C for 15min.
9. Extract once with phenol/chloroform/isoamyl alcohol (50/49/1) and once with chloroform/isoamyl alcohol (49/1). To supernatant, add 180µl 7.5M NH₄Ac and vortex. Add 1ml ethanol down the side of the tube slowly so an interface forms. Swirl tube and then vortex. Spin at RT for 10min. Take off supernatant and wash pellet with 1ml 70% ethanol. Dry for 5min. Redissolve in 200µl TE and use 5µl per digest on 0.7% gel.

Riboprobe labeling is described here

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