MOBIO\CSCLDNA

4/7/94

 

LARGE CsCl PURIFIED DNA PREP

 

A)GROW UP A LARGE FLASK OF CELLS EXPRESSING YOUR PLASMID:

 

1.Make up 500 ml or 1 liter of LB media (10 g NaCl, 5 g Yeast Extract, 10 g Tryptone in 1 liter of deionized water- autoclave in flask), supplement with Ampicillin to 100 mg/liter (4 ml of 25 mg/ml stock into 1 liter).

 

2.Add 5 ml of overnight culture or about 10 ul of frozen stock, or something similar with bacteria containing your plasmid.Shake overnight at 37o C.

 

 

B)HARVEST BACTERIA AND MAKE CRUDE DNA (for 500 ml cells, split to two for 1 liter):

 

3.Pellet the cells in 250 ml bottles in the Sorval superspeed centrifuge (10 minutes at 8 krpm).Pour off the medium.

 

4.Resuspend the bacterial pellet in 10 ml of miniprep solution 1.Vortex in 250 ml bottle to resuspend and keep them in bottle at room temp 5 minutes.

 

5.Add 20 mls of miniprep solution 2 (ice cold, freshly prepared 1% SDS, 0.2 N NaOH).Invert the bottle several times to mix.Be gentle.Do NOT vortex.Incubate on ice 15 minutes.

 

6.Add 7.5 ml of 3 M NaOAc, pH 5, invert gently to mix.Leave on ice 60 minutes.

 

7.Spin crap out 10 minutes 10 krpm.Pour supernatant into new 250 ml bottle, add 20 ml isopropanol (2-propanol).Incubate 20 minutes at room temperature.

 

8.Spin 10 minutes at 8 krpm, pour out the supernatant, and resuspend the pellet in 8 ml TE (10 mM Tris pH 8.0, 1 mM EDTA) by pipetting.Transfer resuspended pellet into 50 ml round bottom superspeed centrifuge tube, add 4 ml of 7.5 M NH4OAc.Incubate on ice 20 minutes.

 

9.Spin out crap at 8 krpm for 15 minutes.Transfer the supernatant to a new tube and add 24 ml EtOH.Incubate on ice 20 minutes.

 

10.Spin out crude DNA 10 krpm, 15 minutes.Pour off supernatant.Resuspend the pellet in 4.5 ml TE (if you are doing a double prep, combine the two pellets at this step).

 

11.To the DNA/TE solution add 4.6 g CsCl and dissolve.Add 0.5 ml 10 mg/ml Ethidium Bromide solution, mix.

 

C)FIRST CESIUM GRADIENT:

 

12.Measure 5 ml of a sterile CsCl:water (1:1) solution into a 15 ml conical tube and transfer this into a quickseal ultracentrifuge tube using a sterile pasteur pipette fitted with a rubber bulb.

 

13.Carefully overlay your DNA solution containing CsCl and Ethidium Bromide using another sterile pasteur pipette.

 

14. Fill the tube with paraffin oil using pasteur pipette/bulb.Seal tube with heat sealer in the mol core.

 

15.Place tube and balance tube in Ti70 rotor, place red caps (next to heat sealer in mol core) on top of each tube.Spin overnight (20 hours) at 50 krpm at 15o C in ultracentrifuge.

 

 

D)SECOND CESIUM GRADIENT:

 

16.Bring down ultra (no brake).Remove rotor, pull out tubes with samples (try not to disturb the gradient; a hemostat is useful).The DNA band should be visible in the tube- it is darker red.There will also be lots of crap in the tube:Don't worry about it.Place tube in a clamp on a ring stand and adjust it to your eye level (sit on a low stool).

 

17.Poke one 18 ga. needle into the top of the tube to allow air to enter the tube.Poke another 18 ga. needle, this one with an attached 3 or 5 ml syringe into the tube about 0.5 cm below the bottom of your band.With the flat part of the bevel pointed up, slowly draw the DNA band into the syringe, tryin to minimize disturbing the band, and minimizing recovery of CRAP.Put the DNA into a 15 ml conical tube.

 

18.Using same techniques as on first gradient, put 5 ml CsCl:water (1.6 g: 1 ml) into a quick seal tube, layer DNA solution on top, and layer 4 ml of CsCl:water (0.5:1.0) on top.Fill with paraffin oil and seal.Spin again overnight (15+ hours) at 50 krpm in ultra.

 

E)RETRIEVE AND CLEAN UP THAT DNA:

 

19.Pull the DNA band as before, transfer it into a 15 ml conical tube, and remove the Ethidium Bromide by extracting at least three (3) times with CsCl saturated isopropanol.(CsCl saturated isopropanol:Mix equal volumes of isopropanol and 0.9 g/ml CsCl solutions, allow to separate.Isopropanol is the upper phase).Remove the isopropanol as completely as possible.

 

20.Add 3 volumes of water.

 

21.Add 2 volumes of EtOH (this means 2 x (vol of original DNA soln + water) not 2 x original DNA volume) and incubate at -20o C for 2 hour to overnight.

 

22.Pellet the DNA by spinning for 15 minutes at 8 krpm.Pour off the supernatant and dry the pellet briefly.

 

23.Resuspend the DNA pellet in 300 ul TE.

 

(For Transfections complete the following steps, as well):

 

24.EtOh ppt the DNA by adding 1/10 vol 3 M NaOAc (pH 7), and 2 vol EtOH. (15 minutes room temperature).Spin down the DNA pellet.

 

25.Wash the pellet in 70% EtOh, dry

 

26.Resuspend in TE.

 

 

 

 

AT THE END OF THE PROCESS:

 

Estimate DNA purity and concentration by reading OD260 and OD280.

-- Purity: 260/280=2

--Concentration:OD260 x50 ug/ml-OD.Donít forget dilution factor...