Single-stranded DNA preparation for sequencing

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Optimized for ssDNA from phagemid pBluescript propagated in E.coli host XL1 (Stratagene).

Triton Method: For manual sequencing with T7 polymerase and 35S-labelled ddATP, or cycle sequencing with Thermosequenase (Amersham), in combination with electrophoresis on the automatic sequencer Licor 4000L.

SDS Method: For cycle sequencing with Sequiterm (Epicentre) (or Thermosequenase (Amersham)), in combination with electrophoresis on the automatic sequencer Licor 4000L.

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0. MEDIA AND REAGENTS--------------------------------------------------------------------------------LB stock: 5x, filter sterilized, frozen.Kanamycin stock:10 mg/mlAmpicillin stock: 100 mg/mlChloramphicol stock:25 mg/mlTetracycline stock: 5 mg/mlCulture medium: LB 1/2 xM9 salts 1 xGlycerol 1%Tetracycline 5 ug/mlAmpicillin 100 ug/ml. PEG-solution: PEG Carbowax-8000 (20%);Ammonium acetate (3.5 M), pH 7.5.Triton stock: 10 % (store at -20oC)Proteinase K stock:10 mg/ml (store at -20oC)Triton/Prot.K-soln :Triton 0.1 %Tris 100 mM, pH 7-8 EDTA 5 mMProteinase K 100 ug/ml. Mix freshly together before usage.Helper phage K07 stock:10exp12 pfu/ml stockCulture containers: Cell Well culture plates (Corning 25820).---------------------------------------------------------------------------------I. CULTURESThe day before inoculation of liquid cultures, transfer clones onto a fresh agar plate and incubate for 24 hrs at 37oC. For some libraries, if not older than 1 year, clones can be inoculateddirectly from the frozen microtiter plates. 1.Inoculate 100 ul medium, by taking an amount of cells thatcorresponds to a microcolony (size = .). 2.Shake cultures at 37oC for 1 hr (=log culture of O.D.600=0.6.) 3.Add 2 ul helper phage (=2x10exp9) and shake for 1 hr (up to 2) at 37oC.4.Add 1.0 ml of LB supplemented with 70 ug/ml kanamycin and shake at 150 rpm for 16-18 hrs (up to 20) at 37oC. II. PREPARATION of ssDNA 1.Pour culture in eppy tube and spin down for 10 min at 13000 rpm. 2.Transfer supernatant into new tube, add 300 ul PEG-soln.3.Let sit for 10-15 min at rt. 4.Spin for 5 min at 13,000 rpm (gives smear or pellet), discard supernatant with suction. 5.Spin again for 1 min and remove carefully the rest of liquid. 6.Add 50 ul Triton/Prot.K-soln, let sit for 5-15 min (rt or 37oC), vortex briefly, incubate at 37oC for 30 min, vortex again.Load an aliquot of 2 ul on an agarose gel to check size and yield(loading buffer must contain 0.2% SDS for better EB staining). Continue incubation for another 30 min. 7.Heat-denature Prot.K for 10 min at 90oC, then chill on ice and spin down for 2-5 min. Take 6 ul of the supernatant for a full sequencing reaction.(It is not necessary to transfer the supernatant to new tube).III. STORAGE of ssDNAFor sequencing purposes, the DNA quality is stable for at least 1 year of storage at +4oC or -20oC. *When stored at 4oC, compensate for evaporation by adding H2O to restore the orignal volume. Before using the DNA for a sequencing reaction, mix the DNA solution, spin turbitidy down briefly and take an aliquot from the supernatant. *When stored at -20oC, repeat step II.7 before using the DNA fora sequence reaction.

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0. MEDIA AND REAGENTS--------------------------------------------------------------------------------LB stock: 5 x LB, filter-sterilized (stored at -20oC)Ampicillin stock: 100 mg/ml (in H2O)Tetracycline stock: 5 mg/ml (in 50% ethanol)Kanamycin stock:70 ug/ml (in H2O)Culture medium: 1/2 x LB, 1 x M9 salts, 1% glycerol, 100 ug/ml ampicillin, 5 ug/ml tetracycline.Culture \"tubes\" : Multiwell tissue culture plates Falcon 3047.PEG-soln : PEG Carbowax 8000 (20%), 3.5 M ammonium acetate, pH 7.5.SDS/Prot.K-soln: 0.1 % SDS, 100 mM Tris, 5 mM EDTA, pH 7-8; 100 ug/ml Proteinase K (add Prot.K only before usage.)--------------------------------------------------------------------------------I. CULTURE24 hrs before inoculation, transfer clones onto a fresh agar plate,containting ampicillin and tetracycline.1. Inoculate clones into 100 ul medium, by taking a tiny amount of cells that corresponds to a micro colony (size of this \".\" and not more!).2. Shake cultures at 37oC for 1 hr to obtain a log culture of ~O.D.600=0.6.3. Add 2x10exp9 helper phage K07 (2 ul of 10exp12 pfu/ml stock)and shake at 37oC for 1 hr at 150 rpm.4. Add 1.0 ml LB supplemented with 70 ug/ml kanamycin and shake at 37oC foranother 16-18 hrs. Less turbid cultures should be shaken some extra hours. II. PREPARATION of ssDNA1. Pour culture in eppy tube and spin down for 10 min 13000 rpm2. Pour supernatant in new tube and add 300 ul PEG-soln.3. Let sit 10 min at room temperature.4. Spin down for 5 min at 13,000 rpm (gives smear or pellet), discardsupernatant with suction.5. Spin down again for 1 min and remove carefully all residual liquid.6. Dissolve pellet thoroughly by vortexing in 300 ul SDS/Prot.K-soln, incubate at 37oC for 1 hr, or at room temp. o.n.7. Check size of ssDNA on agarose gel (load 5 ul).III. PURIFICATION of ssDNA1. NaCl extraction: add 75 ul NaCl of a 5 M stock solution to ssDNA (gives 1 M final), put for 1 hr on ice, spin for 10 min, turn tube for 180oand spin again for 10 min. Transfer supernatant immediately into new tubes.2. Precipitate ssDNA with ethanol+3M ammonium acetate, put on ice for 10 min,spin for 5 min at 13,000 rpm at 4oC.3. Drip-dry pellet, wash it with 70 % ethanol, drip-dry, then dry forseveral mins at 60oC.4. Redissolve pellet in 12-20 ul TE by vortexing (DNA sticks all over the tube).Yield: up to 10 ug of DNA. Store at -20oC.