Spin down the bacteria from a 100 ml culture in two 65 ml
centrifuge bottles at 5000 rpm for 5 min. in the Hermle. If you have more
than will fit in the two bottles (don't exceed the line on the side of the
tube or you will spray bacteria around inside the centrifuge) spin down what
will fit, pour off the supernatant and then pour the rest in on top of the
pellets and spin again.
Discard the supernatant. Resuspend pellets in 4 ml each
(8 ml total) by vortexing. Pool the solutions into one tube.
Add 16 ml of Solution II, mix gently by rotating the
centrifuge tube slowly. The bacteria should lyse and the
solution become translucent.
Add 12 ml of Solution III, shake to mix. A flocculent
white precipitate should form. You are going for a cottage cheese look. If
you shake too hard your DNA will be contaminated. If you don't shake
enough the yield will be low. Spin 5000 rpm for 10 min. to
pellet the precipitate.
Decant supernatant to fresh centrifuge bottle (65 ml) through
gauze to trap any floating bits of precipitate. Add 0.6 volumes
(25 ml) of isopropanol, mix and spin 8000 rpm for 10 minutes.
Discard supernatant, remove as much as possible by aspiration.
Dissolve pellet in 3 ml TE and transfer to a 12 ml centrifuge
tube (Nalgene PSF tubes or disposable 14ml Falcon tubes). Add 3 ml cold (-20º C) 5M LiCl.
Mix well and spin 5000 rpm for 10 minutes.
Transfer supernatant to fresh 12 ml centrifuge tube, add an
equal volume of isopropanol (7 ml or fill tube - don't overfill it), mix well and
spin 5000 rpm for 10 minutes.
Aspirate the supernatant and leave to air dry.
Dissolve the pellet in 300 ul of TE + 20 ug/ml RNase A;
transfer to a microfuge tube, incubate at room temperature for 15
minutes.
Add an equal volume of 26% PEG in 1.6M NaCl, put on ice for 5
min, mix well and spin 15 minutes at 4ºC in microfuge to pellet
DNA. Wash the pellet with 70% ethanol, speed vac dry before
resuspending in TE. This will remove a lot of the excess PEG,
making the phenol extractions much more bearable!
Dissolve pellet in 400 ul of TE. (Be
sure that the pellet is completely dissolved before proceeding or
your DNA yield may be considerably reduced.) Extract once with
an equal volume of chloroform, twice with an equal volume of
buffered phenol, and once more with an equal volume of
chloroform. Add ammonium acetate to 0.5M plus two volumes of
100% ethanol. Mix by gently inverting the tube several times;
you should be able to see the DNA coming out of solution. Let
stand for 10 minutes at room temperature, spin 5 minutes in
microfuge to pellet the plasmid DNA. PHENOL IS AN ORGANIC ACID. CONTACT WITH SKIN WILL RESULT
IN SEVERE BURNS. WEAR GLOVES, LAB COAT, EYE PROTECTION, AND WORK
IN THE FUMEHOOD.
CHLOROFORM IS A POISON, WEAR GLOVES, LAB COAT AND EYE PROTECTION.
READ MSDS SHEETS FOR THESE MATERIALS BEFORE HANDLING.
ETHANOL IS HIGHLY FLAMMABLE; EXERCISE CAUTION WHEN USING NEAR AN
OPEN FLAME
Aspirate supernatant, wash pellet with 200 ul of 70% ethanol,
vortex and spin to repellet DNA. Aspirate supernatant being careful not to suck
up the pellet! and dry
pellet under vacuum.
Dissolve DNA pellet in 50 to 100 ul of TE, depending on the
size of the pellet. Read OD260 of a 1:100 dilution to quantify
(1 OD unit = 50 ug/ml of dsDNA). Add TE to adjust the DNA
concentration to 1 mg/ml. Store at -20º C.
Note: when using the spectrophotometer, be sure to allow the UV lamp to warm up for at least 15 min before quanitifying your DNA. Failure to do so will result in incorrect DNA concentration.
Use 0.5 - 1 ug of DNA for a restriction digest to map plasmid.
Solution I (Plasmid Prep)
Glucose Buffer
100mL
500mL
0.05M glucose (m.w. 180.16)
0.9g
4.504g
0.025M Tris (pH 8.0)
1.25mL
6.25mL of (2M stock) 0.01M EDTA
2 mL
10mL of (0.5M stock)
pH 8.0 (should be) adjust if not
SOLUTION II (MAKE FRESH) (PLASMID PREP)
1% SDS
5mL of 20% SDS stock
0.2N NaOH
5mL of 4N NaOH stock
make up to 100mL with dH2O
Note: put some water in container first as
NaOH + SDS will ppt!
SOLUTION III (PLASMID PREP)
1000mL
500mL
250mL
3.0M KoAC
294g
147g
73.5g
2.0M HoAC
115mL
57.5mL
28.75mL
Make up to required volume with dH2O
pH 4.8
Keep at -20º C this solution goes off - I
don't know why!
