Cosmid提取技术

 

<center> <b><font> </font><font> </font></b><font> </font></center>

 

Day One

1. 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.

    

2. Grow cultures for 12 hours
   (do not let go for more than 14 hrs.)

    

Day Two

1. Spin down cultures in 250 ml bottles in Beckman JA-17 rotor at 6,000 rpm for 15 min.

    

2. Pour off sups, freeze pellets at -20°C for 10 min.

    

3. 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).

    

   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

    

4. 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)

    

5. Add 7.5 ml 3M NaOAc pH 4.5 to each tube, mix, on ice 10 min to 1hr.

    

6. Spin out bacterial debris in a Beckman JA-20 rotor at 10,000 rpm for 15 min.

    

7. Pour sups to clean tubes, add 14 ml Isopropanol. Let sit at room temp. for 2 Hrs.

    

8. Spin down DNA at 10,000 rpm for 15 min. in JA-20. Pour off sups, drain on paper towel for 5-10 min.

    

9. 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

1. Combine all samples, add 34.8 gm CsCl. Mix on shaker until dissolved.

    

2. 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.

    

3. Fill Beckman Ultracentrifuge tubes, seal and spin at 70,000 rpm overnight.

    

Day Four

1. Break seal on tubes, pull lower bands seen with U.V. light, combine into one tube.

    

2. Extract EtBr with NaCl saturated Isopropanol, extract two times beyond the loss of pink color in the Isopropanol.

    

3. 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

1. 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.

    

2. 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.

    

3. 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.