Cosmid提取技术
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Day One
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Start overnight cultures of Cosmid in fresh L-Broth + 25 ug/ml kanamycin. 4 x 250 ml each, inoculate each with metal loop from frozen glycerol stocks.
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Grow cultures for 12 hours
(do not let go for more than 14 hrs.)
Day Two
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Spin down cultures in 250 ml bottles in Beckman JA-17 rotor at 6,000 rpm for 15 min.
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Pour off sups, freeze pellets at -20°C for 10 min.
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Resuspend in 10 ml GET solution + 1 mg/ml lysozyme each, transfer 5 ml to each of two 30 ml oakridge tubes. (8 tubes total).
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GET Solution
50 mM glucose
25 mM Tris-Cl (pH8.0)
10 mM EDTA (pH8.0)
Make 100 ml batches, autoclave 15 mins, store 4 C -
Add 10 ml of SDS/NaOH solution (1% SDS, 0.2N NaOH) to each tube, mix by gentle inversion 10 times. Lyse on ice for 5 min. (Potential denaturation of supercoils if left longer)
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Add 7.5 ml 3M NaOAc pH 4.5 to each tube, mix, on ice 10 min to 1hr.
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Spin out bacterial debris in a Beckman JA-20 rotor at 10,000 rpm for 15 min.
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Pour sups to clean tubes, add 14 ml Isopropanol. Let sit at room temp. for 2 Hrs.
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Spin down DNA at 10,000 rpm for 15 min. in JA-20. Pour off sups, drain on paper towel for 5-10 min.
- Resuspend in 4 ml/tube TE (10mM Tris-HCl, 1mM Edta) on gentle shaker overnight at room temp. (Have tubes in a tilted rack so the TE covers the pellet.)
Day Three
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Combine all samples, add 34.8 gm CsCl. Mix on shaker until dissolved.
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Add 800 ul of 10 mg/ml EtBr. Weigh 1 ml of solution, add CsCl or TE to adjust the weight of the solution to 1.55 to 1.59 g/ml.
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Fill Beckman Ultracentrifuge tubes, seal and spin at 70,000 rpm overnight.
Day Four
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Break seal on tubes, pull lower bands seen with U.V. light, combine into one tube.
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Extract EtBr with NaCl saturated Isopropanol, extract two times beyond the loss of pink color in the Isopropanol.
- Precipitate the DNA by adding three volumes of H2O, bring up to 0.1M NaCl, and add two volumes of EtOH. Put at -20°C overnight. i.e. 1.25ml cosmid, 3.75ml water, 100ul 5mNaCl, and 10ml EtOH.
Day Five
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Spin down DNA in corex tubes with rubber sleeves at 10,000 rpm in the JA-20 rotor for 15 minutes. Drain off EtOH, invert tubes on paper towels for 5 min.
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Resuspend DNA in 200 ul H2O per tube. Combine two tubes each (400 ul), add 8 ul 5M NaCl (0.1M NaCl final conc.), and 1 ml EtOH. On ice for 15 min.
- Spin down in microfuge for 10 min., remove EtOH, wash with 70% EtOH, spin down 10 min. in microfuge, remove EtOH, air dry 10 - 20 min. on bench top. Resuspend in 400 ul H2O. Take absorbance readings at 260 and 280 nm. of sample diluted 1:20 to 1:50.
YIELD: | < 600mg considered poor |
600ng - 1ug is average. |