Preparation of the Cells

1. Inoculate 30 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate
antibiotic either with a single colony of transformed bacteria or with 0.1-1.0 ml of a small
scale liquid culture grown from a single colony.
To ensure that the culture is adequately aerated :
- The volume of the culture flask should be at least for times greater than the volume of
the bacterial culture.
- The culture flask should be loosely capped
- The culture should be incubate in a rotary shaker set a 250 rpm
2. Incubate the culture at the appropriate temperature with vigorous shaking (250cycle /
minute in a rotary shaker) until the bacteria reach the late log phase of growth ( i.e., an
OD 600 of 0.6)
3. Incubate 500 ml of LN, YT, or terrific broth (pre warmed to 37oC) containing the
appropriate antibiotic in a a 2 liter flask with 25 l of the late log phase culture. Incubate
the culture for 2.5 hours at the 37oC with vigorous shaking (250 cycle / minute on a
rotary shaker)
The OD of the result culture should be -4.0. the growth rate of various bacterial strains or
of a single strain containing different plasmid will vary. Thus, in some case, the culture
may have to be incubate slightly longer than or shorter than 2.5 hours to reach the desired
optical density.
4. Add 2.5 ml of 34 mg/ml chloramphenolic. The final concentration of chromaphenolic in
the culture should be 170g/ml. incubate the culture for a further 12-16 hours at 37oC
with vigorous shaking (250cycle / minute in a rotary shaker).
5. Remove an aliquote (1-2 ml) of the bacterial culture to a fresh microfuge tube and store at
47oC. harvest the remainder of the bacterial cells from the 500 ml culture by
centrifugation at 2700g (4100 rpm in a Sorvall GSA rotor) for 14 minutes at 4oC. discard
the supernatant. Stand the open centrifuge bottle in an inverted position to allow all of the
supernatant to drain away
6. Resuspend the bacterial pellet in 200 ml of ice cold STE. Collect the bacterial cells by
centrifugation as describe in step 5. Store the pellet of bacterial in the centrifuge bottle at
-20oC.
7. Prepare plasmid DNA from the 1-2 ml aliquote of bacteria set aside in step 5 by the
minipreparation protocol (either protocol 1 or 4). Analyze the minipreparation plasmid
DNA by digestion with the appropriate restriction enzyme (s) to ensure that the correct
plasmid has been propagated in the large scale culture
This kind of control may seem overly compulsive. However, it provides valuable
insurance against errors that may be difficult to retrieve and my cause considerable loss
of time.
 8. Allow the frozen bacterial cell pellet from step 6 to thaw for 5-10 minutes at roon
temperature. Resuspend the pellet in 10 ml of ice-cold STET. Transfer the suspension to
a 50 ml Erlenmeyer flask.

Lysis of Cells

9. Add 1 ml of a freshly prepared solution of 10 mg/ml lysozyme.

10. Use a clamp to hold the Erlenmeyer flask over the open flame of a Bunsen burner until
the liquid just start to boil. Shake the flask constantly during the heating procedure.
Warning: during heating, keep the open neck of the flask pointing away from you and
everyone else working in the lab.
11. Immediately immerse the bottom half of the flask in a large (2-liter) beaker of boiling
water. Hold the flask in the boiling water of exactly 40 seconds.
Prolonged exposure of superhelical DNA to heat causes irreversible denaturation. The
resulting cycle coiled DNA cannot be cleaved with restriction enzyme, and it migrates
through agarose gels at about twice the rate of superhelical DNA and stain poorly with
ethidium bromide. Variable amounts of this form DNA are always present in plasmid
prepares by thermal lysis of bacterial lysate is heated uniformly to boiling and that the
lysate is kept at 100oC for exactly the recommended time.
12. Cool the flask in ice cold water for 5 minutes
13. Transfer the viscous contents of the flask to an ultracentrifuge tube (Backman SW41 or
its equivalent). Centrifuge the lysate at 150.000 g (30.000 rpm in Beckman SW41Ti
rotor) for 30 minutes at 4oC.
The denser the growth of the original bacterial culture, the more difficult it is to transfer
viscous lysate to the centrifuge tube. If absolutely necessary, the lysate can be chopped
into chunks of manageable size with a pair of long blade scissors, or it can be partially
sheared by drawing it into the barrel of a 10 ml syringe. This problem generally does not
arise when isolating plamid DNA from bacterial cultures that have been treated with
chloramphenicol.
14. Transfer as much of the supernatant as possible to a new tube. Discard the viscous liquid
remaining in the centrifuge tube.
If the bacterial debris does not form a tightly packed pellet, centrifuge again at 210.000g
(35.000 rpm in Beckman SW4Ti rotor) for a further 20 minutes, and then transfer the
supernatant as describe above
15. (optional) if the supernatant contain visible strings of genomic chromatin or Flocculent
precipitate of protein, filter it though 4 ply gauze before proceeding.

Recovery of Plasmid DNA

16. Measure the volume of supernatant. Transfer the supernatant, together with o.6 volume of
isopropanol, to fresh centrifuge tube(s). store the tube(s) for 10 minutes at room
temperature, after mixing the contents well
17. Recover the precipitated nucleic acid by centrifugation at 12.000g (10.000 rpm in a
Sorvall SS-34 rotor) for 15 minutes at room temperature.
Salt may precipitate if centrifugation is carried out at 4oC.
18. Descant the supernatant carefully, and invert the open tube(s) on a paper towel to allow
the last drops of supernatant to drain away. Rinse the pellet and the walls of the tube(s)
with 70% ethanol at room temperature. Drain off the ethanol, and use a Pasteur pipette
attached to a vacuum line to remove any beads of liquid that adhere to the walls of the
tube(s). place the inverted , open tube(s) on a pad of paper towels for few minutes at
room temperature until no trace of ethanol in visible. At this stage, the pellet should still
be damp.
19. Dissolves the pellet pf nucleic acid in 3 ml of TE (pH 8.0).
20. Purify the crude plasmid DNA either by chromatography on commercial resins (please
see protocol 9), precipitations with poluethylene glycol (Protocol 8), or equilibrium
centrifugation in CsCl-ethidium bromide gradients (protocol 10 dan 11)
21. Check the structure of the plasmid by restriction enzyme digestion followed by gel
electrophoresis.
The typical yield of high copy number plasmid vector or of amplified low cpy number vector
prepared by this method is 3-5 g of DNA/ml of original bacterial culture. The yield of
recombinant plasmid containing inserts of foreign DNA is usually slightly lower, depending
on the size and nature of the cloned DNA fragment. Yield of < 1.0 g/ml indicate that the
plasmid DNA is toxic to E. coli; the complete absence of DNA indicates that the plasmid has
been lost during extraction and purification. The first problem can often be solved by
switching to a low copy number vector and / or to a vector that carries prokaryotic
transcriptional termination signals. The second problem is often the result of accidental loss
of the plasmid DNA pellet after ethanol precipitation. It also occur when recombinant
plasmid is large enough to be precipitated together ewth the chromosomal DNA after boiling.
Such a large plasmid will also be susceptible to breakage during extraction and purification.
This difficulty can solved by using a more gentle procedure of the cell lysis, such as that
described in protocol 7.