Title: Plasmid Extraction, DNA Quantitation and Restriction Digestion

Aim:
Aims for Mini-Prep Procedure for Plasmid DNA Extraction
1. Extract extra-chromosomal DNA from E. coli by alkaline lysis.
2. Handle genetic material without cross contaminating.
3. Properly use the autopipettes.

Aims for Determining DNA Concentration by Fluorescent Quantitation

1. Determine the concentration of plasmid DNA extracts.
2. Have a full understanding of concentration factors

Aims for Restriction digestion and agarose gel electrophoresis of plasmid pBR322
1. Perform restriction endonuclease digestion of plasmid DNA.
2. Set up and run agarose gel of DNA digest
3. Analyse results of the electrophoresed agarose gel
5. Place the tube labelled BstN I to incubate at 60°C and the Hind III tube at 37°C for 1 hr then
store
on ice.
6. Label 3 clean 0.75 ml microfuge tubes pBR322, BstN I and Hind III and add 5 µl loading dye
to
each tube.
7. Place 10 µl of the DNA digest (step 3) in the tubes labelled in step 6. Add 10 µl of undigested
plasmid pBR322 DNA to the remaining tube from step 6.
8. Mix the contents of each tube (DNA and loading dye) by slowly pipetting up and down 2-3
times.
Gel electrophoresis
9. The demonstrators will show you how to load your samples into the agarose gel.
10. The samples will be subjected to electrophoresis at 100V for 30 min in 0.5 x TBE buffer.
11. Carefully slide the gel off the tray and into the staining container.
12. Leave the gel to stain for 5-10 minutes. Destain gel in distilled water for 5 to 20 mins., to
help
remove background ethidium bromide.
13. View and photograph gel under a UV transilluminator
 Results:
Gel electrophoresis of pBR322

1 2 3 4 5 6 7 8 9 10 11 12 13
14141 31313
Negative electrode
Direction of migration

10.0
8.0

6.0 8.0 kb
5.0
4.2 kb
4.0
4.0 kb
3.0
4.0 kb
2.0
3.5 kb
1.5
1.8 kb
1.0
1.0 kb
0.5
0.75 kb

Positive electrode

Key
Lane
8 Undigested pBR322
9 BstNI
10 HIndIII
Table showing fragment sizes for Undigested pBR322 and expected and observed sizes for
HindIII digested pbR322 and BstN1 digested pBR322.
Expected Observed
Molecular Undigested BstN1 HindIII BstN1 HindIII
Marker, 1kb pBR322 digested digested digested digested
Ladder pBR322 pbR322 pBR322 pbR322
10
8 1 fragment
equal to 8kb
6
5
4 1 fragment 1 fragment 1 fragment 1 fragment
equal to 4 kb greater than 4 equal to 4 kb greater than 4
kb kb
3 1 fragment
greater than 3
kb
2 1 fragment 1 fragment
size less than size less than
2 kb 2 kb
1.5
1 1 fragment 1 fragment
size equal to equal to 1 kb
1 kb and 1
less than 1 kb
0.5 1 fragment
greater than
0.5 kb

Sample calculation
expected fragment size.
Fragment 1
1059 -130= 929 bp or 0.929 kb
Gel spotting of DNA

A B C D E F G H
1

A B C D E F G

Row 1 0.5 0.25 0.125 0.125 0.0312 0.0156 0.0078 0.0039

µg/µL µg/µL µg/µL µg/µL µg/µL µg/µL µg/µL µg/µL
 Row 3 Undiluted B8 1/10 B8

Questions
1. Consider the 3 major classes of biologically important molecules: proteins, lipids and nucleic
acid. Which steps in the miniprep procedure act on proteins (4 marks)? On lipids (3marks)? On
nucleic acids (4 marks)?
The 3 biologically important molecules in question which are protein lipids and nucleic acid are
act upon at different steps in the miniprep. Lipids are acted on by the addition solutions 1,2 and
3 which are [TEG (Tris EDTA Glucose) Buffer, TEG + Lysozyme and SDS-sodium hydroxide
respectively. Protein on the other hand is acted upon by SDS-sodium hydroxide, SDS will
denature the chromosomal and plasmid DNA into single strands and the Ammonium Acetate
will precipitate the protein. The nucleic acid is precipitated by both the isopropanol and ethanol
solutions.
2. What other kinds of molecules, in addition to plasmid DNA would you expect to find in the
final miniprep sample (4 marks)?
RNA would be expected to be found in the final miniprep sample because the reagent that was
used to precipitate the other biological molecules does not act on the RNA so it would be
present. RNase H can be used to get rid of the RNA.
4. What do ApR, TcR, and ori on the pBR322 map represent and discuss their functions? (6
marks)
ApR and TcR represent antibiotic resistance genes (selectable markers)
They ensure that plasmid DNA is retained in bacterial populations, and the plasmid contains an
antibiotic resistance gene. ApR gene makes the cell resistant to ampicillin and TcR makes the
cell resistant to tetracycline.

Ori represents the origin of replication.

Plasmids reproduce independently of the bacterial cell cycle due to the origins of replication. The
capacity of the plasmid to be duplicated (amplified) by bacteria is dependent on the origins of
replication, which is one of the reasons why plasmids are so useful.

5. Does the undigested plasmid show more than a single band when electrophoresed? Write
short notes explaining why this may occur. (4 marks)
When the undigested plasmid was electrophoresed it show more than a single band because
Undigested plasmids take up multiple conformations. When uncut plasmid DNA is isolated and
runs on an agarose gel, you are likely to see 3 bands. This is because the circular DNA takes on
several conformations the most abundant being: supercoiled, relaxed and nicked.

Expected & observed band sizes correlate? (5 marks)

The expected and band sizes correlate for the part, however, some of the fragments were
different slightly off for example the HindIII is expected to have one band which is a fragment
greater than 4 kb and this was observed in the electropherogram as there was only one band for
the HindIII Which was 4.2 kb.
Monroe, M. R. (2014, January 14). Plasmids 101: What is a plasmid? Retrieved from

https://blog.addgene.org/plasmids-101-what-is-a-plasmid

Tirabssi, R. (2021, April 19). Plasmid DNA on Agarose gel: The secret of the 3 bands. Retrieved

from https://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-

preps/

Science learning hub. (n.d.). Bacterial DNA – the role of plasmids. Retrieved from

https://www.sciencelearn.org.nz/resources/1900-bacterial-dna-the-role-of-plasmids