Table of Contents

I. Staff involved in the course_______________________________________________________________3

Experiment 1: Preparation of Chemically Competent E. coli Cells __________________________________4
Experiment 2: Bacterial Transformation: The Heat-Shock Method _________________________________6
Experiment 3: Plasmid DNA Isolation________________________________________________________8
Experiment 4: Agarose Gel Electrophoresis for Visualizing Plasmid DNA __________________________10
Experiment 5: Restriction Digest of Plasmid DNA______________________________________________13

2
I. Staff involved in the course

Instructor:
Assist. Prof. Dr. Ilgın Çağnan Çakkol
Room number: AS112
Email address: [email protected]

Laboratory Assistant:
Mustafa Soğancı
Room number: AS117
Email address: [email protected]

Pembe Savaş
Room number: AS114
Email address: [email protected]

Nojan Hafizi
Room number: AS115
Email address: [email protected]

Nermin Türen
Room number: AS120
Email address: [email protected]

3
EXPERIMENT 1: Preparation of Chemically Competent E. coli Cells
Introduction:
Competent bacterial cells are able to accept plasmids (which are extrachromosomal DNA containing the
gene(s) of interest as well as selection and/or antibiotic resistance markers) from the environment. The
generation of competent cells may occur naturally or can be obtained artificially (1, 2). Bacterial cells growing
exponentially can be made competent more straightforwardly than bacterial cells at other stages of growth (2).
The calcium chloride (CaCl2) method prior to heat-shock transformation is used to generate chemically
competent cells (1).
Reference:
1- Chang A.Y., Chau V.W.Y., Landas J.A., Pang Y. (2017). Preparation of calcium competent Escherichia
coli and heat-shock transformation. JEMI Methods, 1: 22-25.
2- How are competent bacterial cells transformed with a plasmid? Retrieved February 27, 2022, from
https://worldwide.promega.com/resources/pubhub/enotes/how-are-competent-bacterial-cells-transformed-
with-a-plasmid/
Reagents:
1. Overnight Escherichia coli (E. coli) culture
2. 0.1M CaCl2 (ice cold)
3. 0.1M CaCl2 with 15% glycerol solution (ice cold)
4. Luria Broth (LB) media
5. Ice
6. 250 mL conical flask
7. Falcon Tubes
8. 10mL serological pipettes
9. Pipette and sterile pipette tips
10. Permanent marker
11. 37◦C shaking incubator
12. Spectrophotometer
13. Centrifuge
Protocol: (adapted from: Chang et al., 2017)
A. Overnight Culture(s): (This step will be carried out by the teaching staff)
• Inoculate 10mL of LB with E. coli
• Place in shaking incubator at 37°C and 150rpm
• Incubate for 12-16 hours

4
B. Generation of Competent Cells (CaCl2 wash)
i. Subculturing overnight culture:
• Add 0.5mL of overnight culture to 50mL of fresh LB (1:100 dilution, no antibiotics)
• Shake incubate at 37°C and 150rpm for 3-4 hours or until OD600 reaches 0.4
ii. CaCl2 wash: (Students will start the experiment from this step)
• Ensure that all reagents (CaCl2 solutions, falcon tubes, centrifuge) are ice-cold or at 4°C
• Transfer 10 mL of the culture into a 15 mL Falcon tube
• Place on ice for 20 minutes
• Centrifuge at 4°C at 4000rpm for 10 minutes
• Discard the supernatant by tipping tubes over a discard bin
• Resuspend each pellet with 5mL ice-cold 0.1M CaCl2, incubate on ice for 30 minutes
• Centrifuge at 4°C at 4000rpm for 10 minutes
• Discard the supernatant
• Resuspend the pellet in 0.5mL ice-cold 0.1M CaCl2 with 15% glycerol
• Use for downstream applications or store in -80°C freezer

5
EXPERIMENT 2: Bacterial Transformation: The Heat-Shock Method
Introduction:
Bacterial cells can be transformed by heat shock or electroporation (1, 2). Depending on which method is used,
the bacterial cells are treated differently in order to make them competent. During the calcium chloride heat-
shock transformation, a plasmid can enter the cell through holes or pores in the bacterial cell wall created by
salt washes and heat treatment (2).
Reference:
1- Chang A.Y., Chau V.W.Y., Landas J.A., Pang Y. (2017). Preparation of calcium competent Escherichia
coli and heat-shock transformation. JEMI Methods, 1: 22-25.
2- How are competent bacterial cells transformed with a plasmid? Retrieved February 27, 2022, from
https://worldwide.promega.com/resources/pubhub/enotes/how-are-competent-bacterial-cells-transformed-
with-a-plasmid/
Reagents:
1. Competent Escherichia coli (E. coli) cells
2. 100ng plasmid DNA
3. 1mL of pre-warmed LB media (at 37◦C)
4. LB agar plates (with appropriate reagent for selection or screening)
5. 100mg/mL Ampicillin (stock solution)
6. Ice
7. 42◦C water bath
8. Centrifuge
Protocol: (adapted from: https://www.addgene.org/protocols/bacterial-transformation/)
1. Transfer the required number of tubes containing competent E. coli cells (DH5α) from -80°C freezer to
wet ice (1 tube per group)
2. Allow the cells to thaw for 5 minutes. Gently tap the tubes multiple times to obtain uniform suspension.
3. Transfer 50µL of competent E. coli cells into a new 1.5mL tube
4. Add 100ng of purified pGLO plasmid DNA (i.e. 1.5 µL) directly to one of the tubes containing competent
E. coli cells. Mix by gentle tapping and place on ice. Then label this tube as ‘+ pGLO’ and with your
initials
a. At the same time, transfer 50µL of competent E. coli cells (without plasmid DNA) to another tube
and this tube should be labeled as ‘- pGLO’ and with your initials
5. Incubate the competent cell/DNA mixture on ice for 20 minutes
6. Transfer the cells to 42°C water bath for exactly 45 seconds
7. Transfer the cells to ice for 2 minutes
8. Add 1mL LB medium (without antibiotics) to each tube (both ‘+ pGLO’ and ‘- pGLO’).
9. Incubates the cells on shaker incubator (150 rpm) at 37°C for 45 minutes

6
10. Pipette 50µL of each transformed cell suspension (‘+ pGLO’) onto LB agar plates containing 50µg/mL
Ampicillin (Amp/LB plate) and spread it using sterile spreader. Incubate plates at 37°C overnight
a. Label each plate with your initials and as ‘+ pGLO’.
11. Pipette 50µL of competent E. coli cells without plasmid DNA (‘- pGLO’) onto a LB agar plate (LB plate)
and spread it using sterile spreader. Incubate plates at 37°C overnight
a. Label each plate with your initials and as ‘- pGLO’.

Taken from: https://www.addgene.org/protocols/bacterial-transformation/

7
EXPERIMENT 3: Plasmid DNA Isolation
Introduction:
A popular technique for obtaining plasmid DNA is known as alkaline lysis (Birnboim and Doly, 1979). This
technique works by separating plasmid DNA from chromosomal DNA using the tiny size and supercoiled
structure of plasmids. It is not meant to be used with complicated samples that contain both bacteria and other
materials; rather, it is only meant to be used with bacterial cultures.
Reference:
Birnboim, H. C., and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid
DNA. Nucleic Acids Res. 7, 1513–1523
Reagents:
1. Buffer 1 (50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 100 µg/mL RNase A)
2. Buffer 2 (0.2 M NaOH; 1 % (w/v) SDS)
3. Buffer 3 (3 M Potassium acetate, pH 5.5)
4. Isopropanol
5. 70% Ethanol
6. Tris-EDTA buffer
7. Spin Columns
8. 1.5 mL microcentrifuge tubes
9. Overnight Escherichia coli (E. coli) culture
10. Luria Broth (LB) media (containing 50µg/mL Ampicillin)
11. 50 mL Falcon Tubes
12. Pipette and sterile pipette tips
13. Permanent marker
14. 37◦C shaking incubator
15. Centrifuge
16. Thermoblock
Protocol: [Adapted from: Anna Behle 2017. Isolation of plasmid DNA from E. coli (Alkaline lysis method).
protocols.io https://dx.doi.org/10.17504/protocols.io.gtbbwin]
A. Growth of Bacterial Cultures (Following steps will be performed by the Laboratory Instructors)
1. Pick a single colony from a freshly streaked selective plate to inoculate 10mL of LB medium supplemented
with the appropriate selection antibiotic (i.e. 50µg/mL Ampicillin).
2. Incubate for 12-16 hours at 37°C while shaking at 150 rpm. Use a tube or flask with a volume of at least 4
times the culture volume.
3. Harvest the bacterial culture by centrifugation at maximum speed for 5 min at room temperature.
4. Decant the supernatant and remove all remaining medium.

8
B. Plasmid DNA Purification
5. Resuspend the bacterial pellet in 350 μL buffer P1.
6. Add 350 μL buffer P2 to lyse the cells. Gently mix by inverting (do not vortex!). Incubate for 5 min at room
temperature. (Note: If incubated for too long, sheared genomic DNA fragments may contaminate the sample.)
7. Add 400 μL buffer P3. Gently mix by inverting (do not vortex!).
8. Centrifuge the samples for 10 minutes at room temperature and maximum speed.
9. Carefully transfer 800 μL of supernatant to a fresh tube, with as little contamination of pelleted material as
possible. If necessary, centrifuge the supernatant again (Step 8) to completely remove the pellet.
10. Add 800 μL of isopropanol. Incubate at -20°C for 10 minutes.
11. Centrifuge sample for 5 minutes at room temperature and maximum speed. Carefully remove supernatant
without discarding the DNA pellet.
12. Wash pellet by adding 500 μL of 70 % ethanol (do not resuspend). Centrifuge sample for 5 minutes at
room temperature and maximum speed.
13. Dry pellet in a thermoblock at 65 ˚C for 10 minutes.
14. Resuspend pellet in 50 μL Tris-EDTA buffer.

9
 EXPERIMENT 4: AGAROSE GEL ELECTROPHORESIS FOR VISUALIZING PLASMID DNA

Introduction:
Gel electrophoresis is a method used to separate DNA, RNA or protein according to its molecular weight, they
are pushed through the gel using an electric current, the gel contains pores that the molecules travels through
depending on the molecular size, the smaller fragments will travel fast and heavier fragments will travel slow
which creates a banding pattern through the gel. When the scientists want to separate DNA and RNA, they
move toward the positive charge of the gel but when they want to separate a protein they have to mix it with
sodium dodecyl sulfate which will unfold the protein and cover it with negative charge. (1)
The gel that is used in the electrophoresis kit has three main times which are the Agarose gel, Polyacrylamide
and starch but we will be focusing on the agarose gel.
Agarose gel is made from the natural polysaccharide polymers extracted from seaweed. Agarose gel is quite
easy to make and they can separate the DNA from 50 base pair to millions of bases. The agarose gel usually
has a percentage between 0.7% and 2% of agarose because when the agarose percentage is higher the longer
the distance between the fragments thus requires more run time can take up to days, in some experiments the
use 3% of agarose for the tiny fragments to make the distance between those fragments longer and easy to
spot. (2)
References:
1- Gel Electrophoresis. Retrieved November 28, 2017, from https://www.nature.com/scitable/definition/gel-
electrophoresis-286
2- Agarose Gel Electrophoresis. Retrieved November 28, 2017, from https://www.addgene.org/protocols/gel-
electrophoresis/
Reagents:
• Electrophoresis chamber and power supply.
• Micropipettes
• Gel casting trays
• Sample combs, which are inserted into the molten agarose to form sample wells in the gel.
• Electrophoresis buffer, Tris-acetate-EDTA (TAE)
• Agarose powder
• A loading buffer, which contains something dense to allow the sample to "fall" into the sample
wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how
far the electrophoresis has proceeded.
• Red Safe, a fluorescent dye used for staining nucleic acids
• Transilluminator (an ultraviolet light box), which is used to visualize stained DNA products in gels.
• Microwave
Preparation:
• In the morning, prepare a gel to be ready
• Plasmid DNA sample
10
Protocol:
- To Prepare Gel;
1. Transfer 100ml of the 1X TAE buffer to a conical flask.
• Make sure the flask is clean.
2. Weigh 0.8 grams of agarose powder (amount depends on the desired percentage of the gel) and add to the
100 ml buffer solution in the flask.
o 0.8% gel will be prepared
o Tip: depending on the size of the bands needed to be separated. A higher percentage agarose
gel will help resolve smaller bands from each other, and a lower percentage gel will help
separate larger bands.
o e.g. 1 g of agarose on 100 mL of TAE will make 1% gel.
3. Microwave the solution until the agarose powder dissolves in the buffer and have a transparent solution
for 1-3 minutes.
• Do not over boil the solution, as some of the buffer will evaporate and thus alter the final percentage
of agarose in the gel.
• Microwave in pulses, swirling the flask occasionally as the solution heats up. HOT. Be careful
stirring.
Tip: it is a good idea to microwave for 30-45 seconds, stop and swirl. –Keep an eye on the solution
4. Take the solution from microwave. Let it cool down to about 50°C, until you can keep your hands on the
flask, for 5 minutes.
5. Add Red-safe to a final concentration, usually about 3 μl of lab stock solution per 100 mL gel.
• Red Safe binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.
• Instead of Red Safe, another option is EtBr which is a known mutagen. Wear a lab coat, eye
protection and gloves when working with this chemical.
6. Pour the solution to a gel tray
• Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the
well comb or towards the sides/edges of the gel with a pipette tip.
7. Place the comb and wait until the gel solidify.
• Place newly poured gel at 4°C for 10-15 mins OR let sit at room temperature for 20-30 mins, until
it has completely solidified.
• If you are in a hurry, the gel will set more quickly if you place the gel tray at 4°C earlier so that it
is already cold when the gel is poured into it.
- Loading Samples and Running an agarose Gel;
8. Add loading buffer to each of your plasmid DNA samples.
• Loading buffer serves two purposes:
1) it provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has
migrated;
2) It contains a high percentage of glycerol that increases the density of your DNA sample causing it
settle to the bottom of the gel well, instead of diffusing in the buffer.
9. Once solidified, Place the gel in the tray in the electrophoretic chamber.

11
10. Pour the buffer solution to the electrophoretic chamber.
• Use the same buffer as in the gel preparation.
• Fill the box until the gel is covered.
11. Carefully load a molecular weight ladder into the first lane of the gel.
12. Carefully load your samples into the additional wells of the gel.
• When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or
buffer from entering the tip.
• Place the very top of the tip of the pipette into the buffer just above the well. Very slowly and steadily,
push the sample out and watch as the sample fills the well.
• After all of the sample is unloaded, push the pipettor to the second stop and carefully raise the pipette
straight out of the buffer.
13. Connect the electrodes and switch on the current.
• A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.
• The DNA is negatively charged and will run towards the positive electrode.
14. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from
the gel box.
15. Place the gel in the UV Transilluminator in order to visualize your DNA fragments.
• The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel.
16. Switch on the Transilluminator and observe the result.
• When using UV light, protect your skin by wearing safety goggles or a face shield, gloves and a
lab coat.
17. Analyze the Gel: Using the DNA ladder in the first lane as a guide (the manufacturer's instruction will tell
you the size of each band), you can infer the size of the DNA in your sample lanes.

12
EXPERIMENT 5: Restriction Digest of Plasmid DNA
Introduction:
Restriction enzyme digestion takes advantage of naturally occurring enzymes that cleave DNA at specific
sequences. There are hundreds of different restriction enzymes, allowing scientists to target a wide variety of
recognition sequences. Restriction enzyme digestion is commonly used in molecular cloning techniques, such
as PCR or restriction cloning. It is also used to quickly check the identity of a plasmid.
Reagents:

• Liquid DNA aliquot of your plasmid

• Restriction enzyme (BamHI)
• Restriction digest buffer
• Thermoblock
• Pipette and sterile pipette tips
• Permanent marker
Protocol: (Adapted from: https://www.addgene.org/protocols/restriction-digest/)
1. In a 1.5mL tube combine the following:

• 0.5 µg DNA (Calculate the volume required to be taken from the stock solution to obtain
0.5 µg DNA)
• 1 µL of Restriction Enzyme (BamHI)
• 3 µL 10x Buffer
• x µL dH2O (to bring total volume to 30µL)
2. Mix gently by pipetting.
3. Incubate tube at 37 °C for 1 hour.
4. To visualize the results of your digest, conduct gel electrophoresis.

13
Experiment 5 (Continued): Analysis of Results by Agarose Gel Electrophoresis
Introduction:
Gel electrophoresis is a method used to separate DNA, RNA or protein according to its molecular weight, they
are pushed through the gel using an electric current, the gel contains pores that the molecules travels through
depending on the molecular size, the smaller fragments will travel fast and heavier fragments will travel slow
which creates a banding pattern through the gel. When the scientists want to separate The DNA and RNA they
toward the positive charge of the gel but when they want to separate a protein they have to mix it with sodium
dodecyl sulfate which will unfold the protein and cover it with negative charge. (1)
Agarose gel is made from the natural polysaccharide polymers extracted from seaweed. Agarose gel is quite
easy to make and they can separate the DNA from 50 base pair to millions of bases. The agarose gel usually
has a percentage between 0.7% and 2% of agarose because when the agarose percentage is higher the longer
the distance between the fragments thus requires more run time can take up to days, in some experiments the
use 3% of agarose for the tiny fragments to make the distance between those fragments longer and easy to
spot. (2)
References:
3- Gel Electrophoresis. Retrieved November 28, 2017, from https://www.nature.com/scitable/definition/gel-
electrophoresis-286
4- Agarose Gel Electrophoresis. Retrieved November 28, 2017, from https://www.addgene.org/protocols/gel-
electrophoresis/
Reagents:
• Electrophoresis chamber and power supply.
• Micropipettes
• Gel casting trays
• Sample combs, which is inserted into the molten agarose to form sample wells in the gel.
• Electrophoresis buffer, Tris-acetate-EDTA (TAE)
• Agarose powder
• Loading buffer, which contains something dense to allow the sample to "fall" into the sample wells,
and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the
electrophoresis has proceeded.
• Red Safe, a fluorescent dye used for staining nucleic acids
• Transilluminator (an ultraviolet light box), which is used to visualize stained DNA products in gels.
• Microwave
Preparation:
• In the morning, prepare a gel to be ready
• Total DNA sample, Fragmented DNA samples

14
Protocol:
- To Prepare Gel;
18. Transfer 100ml of the 1X TAE buffer to a conical flask.
• Make sure the flask is clean.
19. Weigh 1 grams of agarose powder (amount depends on the desired percentage of the gel) and add to the
100 ml buffer solution in the flask.
• 1% gel will be prepared- Explain the difference with other percentages and explain the uses.
Tip: depending on the size of the bands needed to be separated. A higher percentage agarose gel will help
resolve smaller bands from each other, and a lower percentage gel will help separate larger bands.
• e.g. 1 g of agarose on 100 mL of TAE will make 1% gel.
20. Microwave the solution until the agarose powder dissolves in the buffer and have a transparent solution
for 1-3 minutes.
• Do not over boil the solution, as some of the buffer will evaporate and thus alter the final percentage
of agarose in the gel.
• Microwave in pulses, swirling the flask occasionally as the solution heats up. HOT. Be careful
stirring.
Tip: it is a good idea to microwave for 30-45 seconds, stop and swirl. –Keep an eye on the solution
21. Take the solution from microwave. Let it cool down to about 50C, until you can keep your hands on the
flask, for 5 minutes.
22. Add Red-safe to a final concentration, usually about 2-3 μl of lab stock solution per 100 mL gel.
• Red Safe binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.
• Instead of Red Safe, another option is EtBr which is a known mutagen. Wear a lab coat, eye
protection and gloves when working with this chemical.
23. Pour the solution to a gel tray
• Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the
well comb or towards the sides/edges of the gel with a pipette tip.
24. Place the comb and wait until the gel solidify.
• Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until
it has completely solidified.
• If you are in a hurry, the gel will set more quickly if you place the gel tray at 4 °C earlier so that it
is already cold when the gel is poured into it.
- Loading Samples and Running an agarose Gel;
25. Add loading buffer to each of your DNA samples.
Explain: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and allows
you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the
density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer.
26. Once solidified, Place the gel in the tray in the electrophoretic chamber.
27. Pour the 100ml buffer solution to the electrophoretic chamber.

15
 • Use the same buffer as in the gel preparation.
• Fill the box until the gel is covered.
28. Carefully load a molecular weight ladder into the first lane of the gel.
29. Carefully load your samples into the additional wells of the gel.
• When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles
or buffer from entering the tip.
• Place the very top of the tip of the pipette into the buffer just above the well. Very slowly and
steadily, push the sample out and watch as the sample fills the well.
• After all of the sample is unloaded, push the pipettor to the second stop and carefully raise the
pipette straight out of the buffer.
30. Connect the electrodes and switch on the current.
• A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.
• The DNA is negatively charged and will run towards the positive electrode.
31. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from
the gel box.
32. Place the gel in the UV Transilluminator in order to visualize your DNA fragments.
• The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel.
33. Switch on the Transilluminator and observe the result.
• When using UV light, protect your skin by wearing safety goggles or a face shield, gloves and a
lab coat.
34. Analyze the Gel: Using the DNA ladder in the first lane as a guide (the manufacturer's instruction will tell
you the size of each band), you can infer the size of the DNA in your sample lanes.

16
Expected Results:

(Reference: https://www.bio-rad.com/webroot/web/pdf/lse/literature/Bulletin_7433.pdf)

- We expect to visualize three restriction digest products on the agarose gel.

- Total Plasmid Size = 5371bp
o 1st Product= 5371 – (2084-1239) = 4526 bp
o 2nd Product= 1864-1239 = 625 bp
o 3rd Product= 2084-1864= 217bp

17