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content/Biokemi/Plasmidlabb/Plasmid Lab Part 1 OCR.md

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+Recombinant DNA technology
+Lecture by:
+Anne Wöhr
+anne.wohr@gu.se
+
+Recombinant DNA technology
+Molecular cloning Transformation Selection and 
+Replication
+Performed during this lab
+
+Plasmids
+• Commonly found in bacteria as extra-chromosomal 
+circular dsDNA molecules 
+• Able to self-replicate during cell division
+• Often carry beneficial genes like antibiotic resistance
+• Bacteria can share genetic information through 
+plasmid transfer
+By: Maya Kostman
+How could this be useful for us?
+
+Applications of recombinant DNA 
+technologies
+Khan S., 2016
+Biopharmaceuticals:
+Vaccines eg hepatitis B vaccine
+recombinant proteins eg Insulin (Diabetes), Factor VIII (hemophilia)
+Genetically modified organisms: 
+Organisms that have been genetically modified to exhibit specific traits eg 
+herbicide-resistant crop plants
+Gene Therapy: 
+In some genetic disorders, patients lack the functional form of a gene. Gene 
+therapy attempts to provide a normal copy of the gene to the cells of the 
+patient.
+Gene Analysis: 
+build artificial, recombinant versions of genes that help understand how 
+genes in an organism function
+
+• Scissors: DNA restriction enzymes (DNA digestion)
+• Cutting sites: multiple cloning sites (MCS)
+• Glue: DNA ligase (DNA ligation)
+• Host: bacterial cells (Transformation)
+• Environment: LB medium or LB agar plates (Culturing)
+• Goal: making more identical copies, or expression (making proteins)
+Toolbox for molecular cloning
+
+✓ DNA molecule that acts as a vehicle to carry foreign genetic materials 
+into another cell, where it can be replicated or expressed.
+Vectors
+Ori (origin of replication): 
+Replication is initiated here, enabling the plasmid to reproduce itself.
+MCS (multiple cloning site):
+Short segment of DNA which contains many restriction sites. This
+allows a piece of DNA to be inserted into that region. The used
+plasmid contains a BamHI cleavage site in its MCS.
+AmpR gene: 
+encodes the enzyme beta-Lactamase, which inactivates ampicillin.
+Cells containing a plasmid vector which expresses AmpR can be
+selected from those that do not by growth in an ampicillin-containing
+medium.
+Lac promoter: 
+binding site of RNA polymerase to initiate expression. IPTG binds
+and inactivates the LacI repressor protein and thereby enables
+expression of genes downstream of the promoter.
+LacZ⍺ gene: 
+encodes the alpha-peptide of the enzyme beta-galactosidase.
+Functional beta-galactosidase consits of the alpha- and omega-
+peptide. The used E-coli strain carries the lacZ deletion mutant
+which contains the omega-peptide but lacks the alpha-peptide. The
+activity of mutant beta-galactosidase is rescued by the presence of
+the alpha-peptide present in the plasmid (alpha-complementation).
+
+Restriction enzymes (Scissors)
+✓ Sequence-specific DNA endonucleases
+✓ Recognise and cleave DNA sequences at specific restriction sites
+✓ Generate “sticky end” or “blunt end”
+
+Escherichia coli (Host)
+✓ Model organism in molecular biology
+✓ Gram negative, rod shaped bacteria
+✓ Located in lower intestine
+
+The History of Insulin Production
+1921: Discovery of insulin
+1922: Leonard Thompson became the first person with diabetes ever 
+treated through administration of insulin
+1923: Insulin is commercialized
+Insulin sales kit, Eli Lilly and Company, 1940s
+14 cows or 70 pigs to sustain a diabetic 
+patient for 1 year
+1970: Recombinant DNA technology is 
+developed
+1982: Recombinant insulin is commercialized 
+
+Production of Insulin
+Adapted from “From DNA to Beer: Harnessing Nature in Medicine & Industry”
+
+Purpose of this lab: 
+Determine whether a gene of interest has 
+been successfully cloned into a vector.
+
+Transformation
+
+Transformation
+During the incubation 
+on ice, DNA binds to 
+the surface of the 
+bacterium as a calcium-
+phosphate-DNA 
+complex
+Following a sudden 
+increase in 
+temperature, one or 
+more DNA molecules 
+bound to the surface of 
+the cell is taken up by 
+the competent cell
+
+How can we selectively grow bacteria 
+that have taken up the plasmid?
+
+Selection pressure
+✓ ampR gene encodes for beta-lactamase
+✓ Inactivates ampicilin antibiotics
+✓ Only cells containing vector DNA will grow
+in the presence of ampicilin
+Selection based on antibiotic resistance
+
+How do we select for bacteria with the plasmids 
+carrying the inserts?
+By: Maya Kostman
+
+More detailed info: thermofisher.com
+LacZ gene naturally found in E. coli, encodes β-galactosidase.
+We use an E.coli strain that carries the LacZ deletion mutant which
+contains the omega-peptide but lacks the alpha-peptide and is therefore
+non-functional. The plasmid we use carries the alpha-peptide, rescuing the
+function of mutant beta-galactosidase.
+Blue-white screening 
+
+Lab Schedule
+• Monday (11/24)
+Introductory lecture 12:15 – 13:00
+Lab 13:15 – 14:00 Groups 1-19
+Lab 14:15 – 15:00 Groups 20-38 
+• Tuesday (11/25)
+Lab 11:15 – 11:45 Groups 1-19 
+Lab 12:00 – 12:30 Groups 20-38
+• Wednesday (11/26) Groups 1-19 
+Introductory lecture 8:15 – 9:00
+Lab 09:15 – 16:00
+• Thursday (11/27) Groups 20-38
+Introductory lecture 8:15 – 9:00
+Lab 9:15 – 16:00
+Deadline for submitting lab reports: 07/12/2025
+
+Working in the lab
+✓ Work in groups of 2 people, stick to your assigned partner 
+✓ Always wear gloves and lab coat to protect you and your samples, 
+wash your hands thoroughly before leaving the lab
+✓ When using the pipette, check the volume limits (0.1-10μl, 10-200μl, 
+and 100-1000μl)
+✓ Pipette into the bottom of the tube, do not ”shoot” it (especially when
+working with very low volumes)
+
+Transformation of competent cells
+→ performed during this lab (day 1)
+✓ 1 tube of plasmids (P) (with/without insert)
+✓ 1 tube of competent bacteria (C)
+
+Day 1: Selection of transformed cells
+→ performed during this lab (day 1)
+✓ Pick up bacterial colonies (2 white, 2 blue) from plate
+✓ Grow in LB medium with antibiotics (expand the colony and replicate plasmids)
+Day 2: Picking & expansion of blue and white
+colonies
+→ performed during this lab (day 2)
+
+Day 1: Materials for Transformation
+Falcon tube (50ml) LB-agar plate Eppendorf tube 
+Day 1: Spread plate method
+o Apply light pressure to not tear or stab the agar
+
+Day 3 - work overview
+✓ Purify plasmids 
+✓ Restriction enzyme digestion
+✓ Run on agarose gel
+✓ Interpret results
+
+Lab reports
+✓ Write according to the guidelines on the handout on Canvas
+✓ One lab report per group (your names and project group number on 
+the cover page)
+✓ In English
+✓ Upload your lab reports on CANVAS deadline on 07/12/2025
+
+Lecture Questions
+1. Name three applications of molecular cloning.
+2. What is a plasmid?
+3. Name the three main steps involved in recombinant DNA technology?
+4. What is a restriction enzyme?
+5. What are the bacteria used in your protocol?
+6. Explain the calcium/phosphate (heat-shock) method and what it is used for.
+7. How can we selectively grow bacteria that have taken up the plasmid?
+8. Explain the principle behind blue and white screening and its purpose in this
+lab.
+
+References
+Kehoe A (1989). "The story of biosynthetic human insulin". In Sikdar SK, Bier M, Todd PW (eds.).
+Frontiers in Bioprocesssing. Boca Raton, FL: CRC Press. ISBN 978-0-8493-5839-5.
+https://www.sigmaaldrich.com/SE/en/technical-documents/technical-article/genomics/cloning-and
+expression/blue-white-screening
+https://www.nlm.nih.gov/exhibition/fromdnatobeer/exhibition-interactive/recombinant
+DNA/recombinant-dna-technology-alternative.html
+Khan, S., Ullah, M. W., Siddique, R., Nabi, G., Manan, S., Yousaf, M., & Hou, H. (2016). Role of 
+Recombinant DNA Technology to Improve Life. International journal of genomics, 2016, 2405954. 
+https://doi.org/10.1155/2016/2405954
+https://www.thermofisher.com/se/en/home/life-science/cloning/cloning-learning-center/invitrogen
+school-of-molecular-biology/molecular-cloning/cloning/traditional-cloning-basics.html
+

content/Biokemi/Plasmidlabb/Plasmid Lab Part 2 OCR.md

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+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.