MCBI DNA Science Practical

Extraction, Purification and Analysis of plasmid DNA

Aim: To isolate plasmid DNA from bacterial cells, and analyse the extract by gel electrophoresis

Learning Outcomes:

1. Extract and purify DNA by microisolation

2. Analyse DNA by agarose gel electrophoresis

The presence of a plasmid in a bacterial cell may be detected phentotypically, however, it is often
desirable to isolate the plasmid DNA from the host cell for molecular studies. There are a number of
different methods for isolating purified plasmid DNA. The most commonly used small-scale method is
that of Birnboim & Doly and is called the alkaline lysis method. This method can be used to screen a
large number of individual bacterial isolates at the same time which is very important in a cloning
experiment when transformed colonies must be examined for inserts in their plasmids as well as the
insert size.

Alkaline Lysis Method by Microisolation

Solutions

1. 50mM glucose, 25 mM Tris-HCI, pH 8.0, 10 mM EDTA containing 4 mg cm -3 lysozyme

2. 0.2 M NaOH, 1% SDS

3. 3 M potassium acetate, pH 4.8

Protocol

1. You are provided with the following cultures :- (1) E.coli - JA221 + pBR328
(2) E.coli - JA221 + pBR328
with insert 44

2. Take the Eppendorf tubes with culture (each holds 1.5 cm3) and centrifuge the tubes
for one minute in the microfuge at full power.

3. Discard the supernatants (liquid part) without disrupting the pellets and remove as
much of the liquid media as possible.

4. Add 100 μl of ice-cold solution 1 to the pellets. Vortex to completely resuspend.

5. Leave the tubes on the bench (at room temperature) for exactly five minutes.

6. Add 200 μl of solution 2. Mix vigorously by rapidly inverting the tubes several times.

7. Leave on ice for exactly five minutes.

8. Add 150 μl of ice-cold solution 3. Gently mix by slowly inverting the tubes ten times.

9. Leave on ice for exactly five minutes.

10. Centrifuge the tubes for five minutes in the microfuge at full power.
 11. Remove the supernatant carefully from each tube and pipette into a fresh tube.
Discard the tubes containing the pellets.

12. Estimate the volume of each solution and then add two volumes of absolute ethanol
to each tube and mix by vortexing.

13. Leave on the bench (at room temperature) for two minutes.

14. Centrifuge the tubes for five minutes in the microfuge at full power.

15. Remove the supernatant and invert the tubes over a paper towel to drain off the
ethanol for 5 mins.

16. Wash the pellets with 1 cm3 of 70% ethanol. Flick the tubes with your finger several
times and then re-centrifuge at full power for 1 minutes.

17. Remove the supernatant and invert the tubes on a paper towel to drain off the
majority of the ethanol and allow the pellets to air dry.

18. Add 15 μl of TE to each tube to resuspend the DNA.

19. Add 5μl stopping mix to each tube and mix well.

20. Separate the samples by agarose gel electrophoresis as directed below

Agarose Gel Electrophoresis

Prepare an 0.8% agarose mini-gel as follows. (Molten agarose is prepared for you).

1. The agarose gel is cast in a perspex gel former. Enclose the open sides with tape if it has
not been done so already.

2. Place the sample well forming comb about 1.5 cm from one end of the gel former.
(Note: Ensure that it slots into position.)

3. Pour the agarose into the gel former quickly and allow to set for 10 minutes, ensure 3ul of
GelRed or equivalent dye is added. Wash out the flask that contained the agarose with tap
water.

4. After the gel has set carefully remove the comb and tape and place the gel former in the
main mini-gel apparatus.

Gel Loading and Running

1. Add gel-running buffer (GRB) to the chamber to cover the gel and flood the wells.

2 Carefully pipette 15ul of the samples into the wells and note the loading order, if a DNA
marker (DNA ladder) is available load this into an adjacent well.

3 Place the lid on the chamber and connect the electrodes to the power supply.

4 Run at 130V (constant voltage) until the blue dye is about halfway down the gel.

5. Turn off the power supply, remove the gel and place in the box provided

6. The gel can be viewed on the DNA transilluminator using a UV source.