Alkaline Lysis and CsCl Purification of BAC DNA for Transgenic Production.

This method was contributed by Michelle Southard-Smith and Ron Chandler at Vanderbilt University, Nashville, Tennessee.

This procedure has been derived from a combination of methods used to isolate high MW DNAs.  The lysis conditions and CsCl steps were incorporated from methods used in Ray  MacDonald’s lab at UT Southwestern Medical Center.  The step 10 that uses 7.5M K-acetate is designed to remove contaminating E coli chromosomal DNA and derives from  protocols used at the Washington University School of Medicine, Genome Sequencing Center.  The rest we pieced together from lots of lab experience and lengthy discussions with others making BAC transgenics like Doug Mortlock.  We hope it is helpful to investigators in the field attempting to make transgenic mice with BAC clones.
 
 

Special Considerations
Never vortex, only rock slowly or swirl to mix.
When redissolving DNA pellets, place tubes on a flat rotator for about 15 minutes at room temperature.



1.  Inoculate 2.5ml of LB (+ antibiotic) with single colony or directly from glycerol stock., Let grow overnight shaking at 37°C.

2.  Use 1ml  of overnight culture to inoculate 500ml of LB (+antibiotic) x 2 for a total culture volume of 1L, shake overnight (about 16hrs) at 37°C.

3.  Collect cells by centrifugation in 250ml x 4 Nalgene bottles, (H-6000A rotor, 3800 rpm), 10 minutes, 4°C.

4.  Wash cells by resuspending in 25 mls of ICE COLD 10mM NaCl (0.1ml 5M into 50ml final) per 250 ml bottle, pellet at 3800 rpm for 10minutes at 4°C.

5.  Decant supernatant, (freezing the pellet at minus 80°C overnight at this step increases cell lysis efficiency), resuspend pellet from each 250ml culture in 10 ml of Solution I by pipeting up and down with 10ml stripette:  Let stand for 5 minutes at room temperature.

Solution I     10mM EDTA, pH 8.0
for 100ml bring up  2ml of 0.5M EDTA, pH 8.0 to 100 ml

6.  Add 20ml of SolutionII per each 250ml of culture, swirl to mix (DO NOT INVERT) and let stand for 5 minutes at room temperature.

Solution II (make up fresh EVERY time)  for 50ml
0.2M NaOH       1ml of 10N NaOH (20g NaOH into 50mls for 10N)
1% SDS             5mls of 10% SDS (50g into 500mls for 10%, filter sterilize)

7.  Add 15 ml of ice cold solution III per each 250ml of culture, mix gently by swirling and place on ice for 10 minutes.
(Note: To insure proper neutralization, you may want to keep on ice for longer than 10 minutes.  Clontech’s Nucleobond AX-500 protocol calls for  30-40 minutes.  We keep on ice for minimally 20 minutes.)

Solution III
K-acetate (7.5M Stock, formula weight 98.15, use 184.03g into 250ml autoclaved MilliQ H2O, store at room temperature) 50ml
Glacial Acetic Acid          23ml
Water            127ml

8.  Spin at 15,300 xg RCFMax (10,000 rpm Beckman JA14 rotor; 10,000 rpm Sorvall SLA-1500 rotor) for 15 minutes at 4°C, remove supernatant, then repeat spin on supernatant in a fresh bottle.
(Note: You may want filter supernatant through a pleated Whatman #1  filter paper using autoclaved funnels.)

9.  Add 45 ml of isopropanol to the supernatant and mix.  Spin at 3850xg RCFMax (5,000 rpm Beckman JA14 rotor;  5,000 rpm Sorvall SLA-1500 rotor) for 15 minutes.

10.  Pour off the supernatant and dissolve pellet in 9 ml of 10T/50E (10mM Tris-HCL pH 8.0 (0.5ml of 1M); 50mM EDTA pH 8.0 (5ml of 0.5M)) then transfer to an Oakridge tube (aka SS34).
Add 4.5 ml of K-acetate (7.5M), mix gently,  and keep at minus 80°C for 15-30 minutes.

11.  Thaw completely (avoid mixing) and spin at 3,000xg RCFMax (5,000 rpm Beckman JA20 rotor;  4,500 rpm in Sorvall HB-6 swing rotor) for 10 minutes.
Combine supernatants into a 50 ml conical tube (Note: You should have a total volume of 54 mls at this step.)
Split supernatant by transferring 9 mls into 6 akridge tubes.  Add 22.5 ml of 100% Ethanol to each tube, mix gently.

12.  Pellet DNA at 3000xg RCFMax (5,000 rpm Beckman JA20 rotor;  4,500 rpm Sorvall HB-6 rotor) for 10 min and redissolve precipitate in 1.3ml of 50T/50E (50mM Tris-HCL pH 8.0 (2.5ml 1M Tris-HCL); 50mM EDTA pH 8.0 (5.0 ml 0.5M EDTA)). Pool volumes x 3 into a 2059 Falcon tube (Note: You should have 2  2059 Falcon tubes with 4 mls in each at this step.)

13.  Extract aqueous with 2 ml of phenol (tris-buffer saturated) by gently rocking, let sit for 3 minutes.
Add 2 ml of CHCl3, mix by gently rocking, then separate phases by 3-5,000 rpm in HB-6 swing rotor.

14.  Remove aqueous layer (~ 4 ml) with  wide-bore 2ml stripette (break the end off the 2ml plastic pipette to make it wide-bore) into Falcon 2059 tube.
Add 1/10 (400 ul) volume of 3M NaOAc pH 7.0.
Add 2.5 volumes of 100% Ethanol (~10 ml), mix gently by inversion and place at minus 20°C for at least 30 minutes, preferably 2 hours.

15.  Pellet the DNA by centrifugation at 6000 rpm in swinging bucket Sorvall HB-6 rotor at 4°C for 15 minutes.

16.  Dissolve pellet in 2 ml 50T/25E (50 mM (2.5 ml of 1M) Tris-HCL pH 8.0, 25 mM (2.5 ml of 0.5M) EDTA pH 8.0).
Once dissolved, combine volumes into one 2059 Falcon tube (total of 4 ml).
Add 4.8 g CsCl, dissolve by gentle rocking/warming (in your hand).
When completely dissolved, add 0.4ml EtBr.  (from this point PROTECT FROM LIGHT to avoid nicking of the DNA)

17.  Spin at 3000 rpm in swinging bucket rotor to remove precipitated protein, pour supernatant into new tube avoiding protein scum.
Transfer supernatant to ultracentrifuge tube. (Sorvall # 03945, Tube PA, 6ml volume)  Density ~1.6g/ml on gradients.

18.  Balance tubes to within 0.1g, mount in Sorvall TV865 rotor, tighten caps to 20 lbs with the torque wrench.  Ultracentrifuge overnight at 55,000 rpm, 20°C, zonal 4, NO brake on deceleration..

19.  Insert 25-30g needle into top of tube to provide pressure outlet, pull bands with 18g needle and 1ml syringe to pull the BAC band (Supercoiled BAC DNA is the lower band).
Remove band in the smallest possible volume (should be < 1 ml).

21.  Extract EtBr with TE pH 7.5/100mM NaCl-saturated butanol (1:1 volume ratio), >5 times, or at least 1 extraction past step where all orange color is removed.
Note:  to make TE/NaCl saturated butanol, make up 10mM Tris pH 7.5, 1mM EDTA, 100mM NaCl then add to butanol with intermittent mixing until two phases are observed (butanol is less dense and will be the upper phase).

22. Dialyze the DNA using three changes of 1X Polyamine Microinjection buffer (1 liter volume per change) at 4°C spinning very slowly in baked glassware to avoid nucleases.
We make up the polyamine buffer, sterile filter it and then use it immediately for dialysis.
Place dialysis membrane into new beaker every 4 hours with final overnight dialysis.
1X Polyamine buffer consists of:  10 mM Tris, pH 7.5, 0.1 mM EDTA, 30 microM spermine, 70 microM spermidine, 100 mM NaCl

For 1 liter:
 10mM Tris 7.5 use 1ml of 1M Tris-HCl, 7.5 stock (autoclaved)
 0.1mM EDTA  use 20 ul of 0.5M EDTA, 8.0 (autoclaved)
 10mM NaCl  use 2ml of 5M NaCl stock (autoclaved)
 1X PolyAmines use 100 ul of 1000X PolyAmine Stock
(30mM Spermine, 70mM Spermidine)
 MilliQ H2O up to final volume of 1000ml and then filter sterilize

23.  Take an aliquot after Dialysis and evaluate concentration by A260 to obtain a rough estimate of yield.  At this point concentration is low enough that it's best to read 100ul of dialysate directly in the cuvette and then discard.  Use leftover dialysis buffer for blank on A260. Store BAC DNA at 4C.

24. When you submit your BAC DNA, also submit 10 microliters of Not I digested BAC DNA for pulsed field analysis. This will allow us to assess the purity of your DNA preparation. We will verify the concentration of your concentrated DNA sample and adjust it for microinjection to 0.5 to 1.0 ng per ul.

 
Dialysis Membrane Preparation

Use Spectra/Por dialysis tubing (#132 650) 6-8,000 MWCO.

Boil 15 min in 0.1M NaHCO3 on a hot plate in a glass beaker (use another glass beaker sitting on the membrane to weigh it down, include white boil-easer chips)

Rinse well in milliQ or double distilled H20

Boil 15 min in 0.02M EDTA

Rinse well in milliQ or double distilled H20

Store in 70% ETOH at 4 C

Rinse well in milliQ or double distilled H20 before use to remove ETOH and then rinse with dialysis buffer.

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