Biology Reference
In-Depth Information
Table 5.7: Small-Scale Plasmid Preparations (Minipreps).
1. Prepare 5-ml transformed
E. coli
cultures in LB broth containing the appropriate antibiotic. The cultures
can be grown in disposable 14-ml plastic centrifuge tubes by picking colonies with a sterile toothpick
and dropping the toothpick into the tube. Cap the tube and incubate at 37°C with shaking (250 rpm)
for 16 hour.
2. Pellet the cells by centrifugation at 5000 rpm for 5 minutes. Discard supernatant and toothpick.
3. Add 100
μ
l of 50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 2 mg/ml lysozyme (freshly
prepared). Resuspend pellet and incubate for 10 minutes.
4. Add 200
μ
l 0.2 N NaOH, and 1% SDS. Mix gently. Incubate on ice for 10 minutes. The SDS-NaOH
solution must be made just before use and kept at room temperature. Mix 3.5 ml of water, 1 ml of 1 N
NaOH, and 0.5 ml of 10% SDS.
5. Add 150
μ
l of 3 M potassium acetate (pH 4.8). Mix gently. Incubate for 10 minutes in a freezer. A white
precipitate will form.
6. Centrifuge for 15 min at 15,000 rpm at 4°C.
7. Pour supernatant into Eppendorf tubes and fill with cold ethanol. Incubate in ice for 10 minutes.
8. Pellet the DNA for 1 minutes in a microcentrifuge and aspirate off supernatant. Add 0.5 ml of cold 70%
ethanol and aspirate off.
9. Dry under vacuum. Resuspend in 50
μ
l of distilled water containing 10
μ
g/ml pancreatic ribonuclease
(RNase) to remove RNA.
10. The DNA is suitable for restriction analysis or fragment preparation. Use 5-10
μ
l per reaction. If the
DNA does not cut well it can be re extracted with phenol and precipitated with ethanol.
molecular weight RNA to precipitate. If the pH of the alkaline denaturation step
has been controlled carefully, the plasmid DNA molecules will remain circular-
ized and in solution, but the contaminating molecules will precipitate. The pre-
cipitate can be removed by centrifugation and the plasmid can be purified and
concentrated by ethanol precipitation (
Table 5.7
).
However, many kits are now available that can be used to extract plasmids
from transformed
E. coli
, and automated work stations even are available for
laboratories with a high-volume workload.
5.12 Electrophoresis in Agarose or Acrylamide Gels
DNA and RNA molecules can be separated by size and visualized by agarose or
acrylamide gel electrophoresis.
Gel electrophoresis
provides a powerful method
for resolving mixtures of single- or double-stranded (ss or ds) nucleic-acid mole-
cules. The nucleic acids can be visualized
in situ
in the gel by staining with ethid-
ium bromide (EtBr) and examining it under UV light (
Figure 5.5
).
At a pH near neutrality, linear DNA is negatively charged and migrates from
cathode to anode in a gel, with its mobility dependent on fragment size, volt-
age applied, composition of the electrophoresis buffer, base composition, gel
concentration, and temperature.
