medical-notes/content/Biokemi/Plasmidlabb/Slides II.md

Recombinant DNA technology
Lecture by:
Anne Wöhr
anne.wohr@gu.se

Molecular Cloning
→ Performed beforehand (not done during this lab)

Molecular Cloning
→ Performed beforehand (not done during this lab)
We aim to isolate/separate 
these two plasmids to 
select the recombinant 
plasmid and to verify the 
presence of the gene of 
interest

Day 1: Transformation of competent cells
→ performed during this lab (day 1)

Day 1: Selection of transformed cells
→ performed during this lab (day 1 + day 2)

Day 2: Picking & expansion of blue
and white colonies
→ performed during this lab (day 2)

Revision 
Blue-white screening
Plasmid:
• AmpR: Ampicillin resistance (β-lactamase)
• LacZ: α-peptide for functional β-galactosidase 
enzyme
• BamHI restriction site in LacZ gene for 
insertion of DNA
Growth medium:
• Ampicillin: Only successfully transformed 
bacteria carrying plasmids can survive in the 
presence of ampicillin
• IPTG : activates transcription of the LacZ gene 
by binding its repressor
• X-gal: β-galactosidase degrades X-gal. The 
product has a blue colour!

Day 3 - work overview
✓ Purify plasmids 
✓ Restriction enzyme digestion
✓ Run on agarose gel
✓ Interpret results

Day 3: Material
Spin column
Tubes labeled with
A1; A2; A3; A4; H2O
Plasmid preparation kit
Collection tube & 
spin column (blue)

Day 3: Plasmid purification
→ performed during this lab (day 3)
Buffer A1 (Cell Suspension)
Tris/HCl (pH 8.0), EDTA, RNase A
Buffer A2 (Cell Lysis)
NaOH; SDS
Buffer A3 (Neutralization/Binding)
Contains acetate and guanidine 
hydrochloride
Buffer A4 (Wash, reconstituted)
Contains ethanol, NaCl, EDTA, and 
Tris/HCl

cells grown in LB-
media overnight
Transfer 2x 750 µl into 
microcentrifuge tube 
Bacterial pellet = cells + plasmid
→ Discard superatant
Balance the centrifuge
Day 3: harvest cells & purify plasmids
A1 - resuspension buffer
A2 – cell lysis buffer
A3 – neutralization/ 
binding buffer
Plasmid in supernatant
cell debris as pellet
transfer supernatant
to column
Plasmid binding
Discard 
flow-through
Wash with A4
Transfer column to new tube
add H2O to elute plasmid

QIAprep Miniprep Handbook, Appendix A: Background Information, Preparation of cell lysates, p 43 
Plasmid purification
Buffer A1: 
Bacterial cells are resuspended in a buffer containing Rnase A.
Buffer A2: 
Bacteria are lysed under alkaline conditions (NaOH). SDS solubilizes the phospholipid 
and protein components of the cell membrane
Lysis and release of cells contents. Alkaline conditions: denaturation of chromosomal 
and plasmid DNA as well as proteins.
Buffer A3: 
The lysate is neutralized and adjusted to high-salt-binding conditions. The high salt 
concentration causes denatured proteins, chromosomal DNA, cellular debris, and SDS 
to precipitate, while the smaller plasmid DNA renatures correctly and stays in solution. 
DNA is bound to silica membrane of spin columns in high-salt buffer. RNA, cellular 
proteins and metabolites are not retained on the membrane.
Buffer A4: 
Washing and reconstitution of DNA. Salts are efficiently removed by this wash step.
H2O: 
The purified plasmid DNA is eluted from silica membrane by addition of water. The 
elution is pe is dependent on a low salt concentration and a stable pH (pH 7-8.5).

Restriction enzyme working solution:
Restriction enzyme buffer
H2O
Restriction enzyme (keep it cold!)
Add restriction enzyme to a portion of eluted plasmid
KEEP THE REST OF UNDIGESTED PLASMID AS CONTROLS FOR 
LATER USE Incubate at 37°C for 60 min 
Day 3: Restriction enzyme digestion

Day 3: Restriction enzyme digestion
Plasmid without insert (2700 bp)
Plasmid with insert in BamHI site (4200 bp)
Cut with BamHI → bands at 2700 bp and 1500 bp

• Samples are mixed with 6x loading
dye to make them ”heavier” to stay
in wells
• Separation of DNA molecules
based on their size 
• DNA negatively charged
• Agarose gel for separation
• Shorter molecules move faster and 
migrate farther than longer ones
• Visualization of DNA with SYBR 
safe DNA stain
Day 3: Agarose Gel Electrophoresis

GeneRuler 1kb DNA ladder = size marker
marker
2700 bp
1500 bp
4200 bp
Day 3: Expected Results

• Relaxed/linear: intact circle but “nick” in one strand
• Linear: both strands are cut (at the same location)
• Supercoiled: fully intact with both strands uncut, appears in a compact 
form
Plasmid conformation affects migration

Lab schedule
✓ Purify plasmids 
✓ Restriction enzyme digestion
1-2h incubation time → lunchbreak and 
everyone will be back at the same time
✓ Run on agarose gel
approximately 1h → go through the
expected results to be able to ask
appropriate questions
✓ Interpret results
make sure to ask a lot of questions while you have
the chance!!!

Lab reports
✓ Write according to the guidelines on the handout on Canvas
✓ One lab report per group (Names and group number on the cover page)
✓ In English
✓ Upload your lab reports on CANVAS, deadline 07/12/2025

Lecture Questions
1. What are the sites the plasmid contains that allow for this experiment?
2. What are the 3 compounds in the LB plates that allow for selection of
bacteria with plasmid and the distinction of plasmids with and without
the inserted gene?
3. What are the six steps in plasmid preparation and purification?
4. What is a restriction enzyme and why was it used in this experiment?
5. Explain the principle of Gel Electrophoresis, what is it for?
6. What´s the compound that allows for the visualization of the DNA in
the gel?
7. How many times does BamHI cut the plasmid without the insert? And
the plasmid with the insert?
8. Explain the different plasmid conformations that exist.