Basic Instrumental Techniques (BIT)

PRACTICAL SESSIONS 2020

Student's lab notebook

Student name: Group + Subgroup:

Session 1 date: Session 2 date: Session 3 date:

Teacher name and mark: (to be filled by the teacher)

Student's lab notebook instructions:

Lab notebook is only for personal use (each student requires a personal copy).

Pre lab questions are required to be filled individually before the corresponding practical lesson and
the rest of notebook session questions should be filled in the lab during the practical lesson.

Students will be required to explain pre lab questions to the rest of the class.

Lab notebook will be collected at the end of the 3rd practical session for evaluation.

SESSION 1 (P1): Molecular separation using size exclusion column chromatography

SESSION 2 (P2): Protein composition analysis using acrylamide gel electrophoresis with SDS
(SDS-PAGE)

SESSION 3 (P3): Bacterial DNA purification and polymerase chain reaction (PCR)

Cow milk will be used in all the sessions as evaluation sample. Milk analysis is of notable interest
in several research fields as well as agroindustrial and health care areas.

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SESSION 1: Molecular separation using size exclusion column chromatography.

OBJECTIVES

A) Set up and characterize a gel exclusion chromatography.

B) Desalt and characterize a protein mixture using a gel exclusion chromatography.

C) Fractionate a milk sample using a gel exclusion chromatography.

BACKGROUND

Chromatographic techniques are used to separate molecules based on their different size, shape, charge,
solubility and adsorption properties. Exist many types of chromatography, all of them based on the
interactions between three compounds: the sample mixture, the solid phase (stationary) and the mobile phase
(eluent).

Size exclusion chromatography, also known as gel filtration chromatography, is used to fractionate
molecules according to size. The column material is a gel (also called resin or matrix) formed by particles (or
beads) that have small porous (figure 1). Small molecules have access to all the porous of the beads and thus
spend more time within the gel. Large molecules, with diameter much larger than the pore size, will not
penetrate the beads and will be excluded from the gel by passing through the column faster.

Figure 1

Important parameters in gel filtration chromatography:

- The total volume (Vt) of a gel filtration column is the sum of the volume of the solution that get inside of
the gel beads (Vi), plus the volume of the solution outside the gel beads (Vo, also called exclusion volume or
void volume), plus the volume of the gel within the column (Vg). Vt = Vi + Vo + Vg

It can also be calculated by geometry. Vt = Πr2 · h

- The elution volume (Ve) of a compound is the volume of eluent collected until the elution of this
determined compound.

- The exclusion volume (Vo) can be calculated from the elution volume of a compound with larger size than
the pore diameter of the gel beads. This compound cannot penetrate into the bead porous and will be
excluded from the column fast.

The most widely used types of gels are cross-linked dextran, agarose and polyacrylamide (Sephadex,
Sepharose and Biogel, respectively). Depending on their bead pore size, they will be able to separate
molecules of different molecular weight. The upper limit of the fractionation range is the exclusion limit,

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which means that the molecules with molecular weight greater than this will have less probability of
penetrating the gel and will be completely excluded.

In this laboratory activity we will desalt a protein mixture using gel exclusion chromatography with a
Sephadex G-25 gel column. Sephadex (Sepharation Pharmacia Dextran) G-25 gel is the commercial name
for a cross-linked dextran with an exclusion limit of 5,000 daltons (Da). Therefore, molecules with molecular
weight higher than 5,000 Da will be excluded from the column and will elute just after the exclusion volume,
while smaller proteins and salts, which have a lower molecular weight, will penetrate in the gel porous and
elute later.

PRE-LAB QUESTIONS

A. Gel filtration chromatography is widely used. Which are the more relevant applications ?

B. Which are the advantages and limitations of the technique ?

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C. Which elution pattern can we expect if we collect fractions form a protein mixture with inorganic salts ?
Why ?

D. Which elution pattern can we expect if we collect fractions form a skimmed milk sample ? Why ?

MATERIAL AND REAGENTS

- Ring stand and chromatography column - Rack with collection tubes - Calibration mixture
- Glass wool - Graduated tubes - Protein mixture
- Plastic Pasteur pipettes - Beaker for waste - Skimmed milk tube
- Hydrated Sephadex G-25 gel - Elution solution (distilled water)

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PROCEDURE

1. Preparing the gel and packing the column:

- First, weight the required Sephadex G-25 and hydrate it over night at room temperature. Once the gel is
hydrated it can already be packed in the column. (You do not have to perform this step because we have the
gel hydrated and ready to use).
- Place a small amount of glass wool at the bottom of the empty column to avoid hydrated gel to be lost.
- Set the column on a ring stand, with a collected tube bellow (you can use the rack).
- Close the clamp (for regulation of the flow rate).
- Mix the hydrated Sephadex G-25 solution and introduce it into the column using a Pasteur pipette. Avoid
air bubbles. Feel the column to the top.
- Wait until the gel is starting to be separated from the water. Then, open the clamp to elute the extra amount
of water, but always keep some liquid on top of the gel (around 1 cm).
- Add more Sephadex gel and keep eluting the extra water. At the end, 3/4 of the column high have to be full
of sedimented gel.
- Once the column is packed, wash it with 10 mL of elution solution (in our case: distilled water). Avoid
letting column run dry.
- Stop the elution flow by closing the clamp.

2. Calculating the column flow rate:

- Wash column with 10 mL of water. We will use these washes to calculate the column flow rate.
The flow rate is determined by collecting eluant in a 10 mL graduated tube while monitoring the time. Do it
twice. Express the flow rate as a mean of the 2 estimations in ml/min.

Volume Time Flow (mL/min) Flow rate (mean)

Flow
estimation 1
Flow
estimation 2

3. Calibrating the Column:

We first need to know the volumes where molecules of

different size will elute in our column, to later control the
chromatographic separation. For the column calibration we
will use 2 colored compounds with different molecular
weight that will behave as the sample mixture we will
fractionate afterwards. One compound has a higher size than
the gel exclusion limit while the other has a lower size.

Calibration mixture:
- Blue dextran (MW= 2,000,000 Da)
- Potassium dichromate (MW= 294 Da)
Figure 2
To calibrate the column proceed as follows (figure 2):

- Remove the solution from the top of the gel by opening the clamp. When the solution level coincides with
the gel level, close the clamp again.
- Carefully load 500 μL of the calibrating mixture on top of the gel, using a Pasteur pipette.
- Open the clamp to get the sample into the gel. Close the clamp.
- Place a graduated tube below the column. From this moment, we will start collecting fractions.

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- Only when the sample is completely in the gel, add a few drops (5-6) of elution solution to the top of the
column. Not more volume because the sample would be diluted. Open the clamp to get this elution solution
into the gel.
- From this moment, keep adding elution solution to run the chromatography and elute the different
compounds. Control the clamp while you are collecting fractions.
- Collect 4 fractions and annotated the volume of each fraction:

Fraction Characteristics Volume (mL)

Fraction 1 first not colored eluate.
Fraction 2 blue colored eluate.
Fraction 3 not colored eluate in between the blue and yellow fractions.
Fraction 4 yellow colored eluate.

- When the yellow colored compound is completely eluted, wash the column with 10 mL of elution solution.
- Discard the eluate of the collected tubes and wash them.

Draw a schematic graphic of colors and elution volume (mL) of the calibration compounds.

Annotate the elution volumes of the calibration:

Elution volume:
Blue dextran

Potassium dichromate

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How many mL you need to collect from your column to be sure you will collect compounds of 300 Da or
larger?

4. Protein mixture fractionation

Once the column is equilibrated and characterized, we can load the protein mixture to be separated.

Annotate the protein mixture label:

- Fill a tube with 1 mL of water and mark the volume high. Label with this 1mL mark the number of tubs
you determined necessary to collect compounds larger than 300 Da (previous question). Numerate them.
- Load 500 μL of the protein mixture as we did for the
calibration sample. This solution mixture contains one or
more proteins and inorganic salts. Our goal is to get our
proteins desalted.
- Keep adding elution solution to the column and collecting
fractions of 1 mL in the labeled tubes. (figure 3).
- Stop the elution flow by closing the clamp. Avoid letting
column run dry. Figure 3

5. Analyzing protein mixture fractions by measuring absorbance

The presence of proteins in the collected fractions will be determined by spectrophotometry. We will
measure absorbance at 280 nm (where amino acids with aromatic rings absorb) of the collected fractions
from the sample fractionation (step number 3).

- Calibrate the zero absorbance with elution solution.

- Measure absorbance at 280 nm of the collected fractions (only fractions from step 3).

Elution F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
solution
Volume -----
(ml)
OD 280

6. Skimmed milk sample fractionation

Wash the column with 5 mL of elution solution (in our case: distilled water) to clean the column.

Annotate the skimmed milk label:

- Fill a tube with 1 mL of water and mark the volume high. Label with this 1mL mark the number of tubs
you determined necessary to collect compounds larger than 300 Da. Numerate them.

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- Load 500 μL of the skimmed milk as we did for the calibration sample.
- Keep adding elution solution to the column and collecting fractions of 1 mL in the labeled tubes. (figure
3).

7. Analyzing skimmed milk fractions by measuring absorbance

The presence of proteins in the collected fractions will be determined by spectrophotometry. We will
measure absorbance at 280 nm (where amino acids with aromatic rings absorb) of the collected fractions
from the sample fractionation (step number 3).

- Calibrate the zero absorbance with elution solution.

- Measure absorbance at 280 nm of the collected fractions (only fractions from step 3).

Elution F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
solution
Volume -----
(ml)
OD 280

7. All subgroups (SG) results plotting

Protein mixture
Elution F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
solution
SG1 Volume -----
(ml)
SG1 OD 280

SG2 Volume -----

(ml)
SG2 OD 280

SG3 Volume -----

(ml)
SG3 OD 280

SG4 Volume -----

(ml)
SG4 OD 280

SG5 Volume -----

(ml)
SG5 OD 280

SG6 Volume
(ml)
SG6 OD 280 -----

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Skimmed milk
Elution F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
solution
SG1 Volume -----
(ml)
SG1 OD 280

SG2 Volume -----

(ml)
SG2 OD 280

SG3 Volume -----

(ml)
SG3 OD 280

SG4 Volume -----

(ml)
SG4 OD 280

SG5 Volume -----

(ml)
SG5 OD 280

SG5 Volume -----

(ml)
SG5 OD 280

RESULTS AND DISCUSSION

A. Determine the following parameters of your column:

Exclusion volume (Vo):

Inside-beads volume (Vi):

Total volume (Vt):

B. Which is the elution order of the colored compounds? Explain why.

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C. Determine the elution volume of proteins from the protein mixture:

D. Determine the skimmed milk elution profile:

E. Draw a graph (chromatogram) of absorbance intensity versus elution volume (mL) of the protein mixture
(from step 5) as well as skimmed milk (from step 7) . Also mark with an arrow the previously determined
elution volumes of the calibration compounds.

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F. Discuss the obtained results. Consider your data as well as the data obtained with the other subgroups.

SAFETY CONSIDERATIONS

Follow laboratory safety considerations.

Wear gloves and safety glasses when manipulating materials and reagents.

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SESSION 2: Protein composition analysis using acrylamide gel electrophoresis with SDS (SDS-PAGE)

OBJECTIVES

A) Determine the composition of 3 tubes that may contain BSA and gamma globulin by SDS-PAGE using
reducing and non-reducing conditions.

B) Visualize raw cow milk +/- casein protein profile by SDS-PAGE using reducing conditions.

BACKGROUND

SDS-PAGE (Sodium Dodecyl Sulfate -PolyAcrylamide Gel Electrophoresis) is a widely used technique as it
provides large amount of information and it is a flexible technique.

Several modifications from the basic technique have been developed. Nevertheless running gel and loading
buffer composition are probably the most relevant parameters to take into account when analyzing a SDS-
PAGE results:

Running gel composition: Acrylamide concentration can range from 7.5 to 15% (it depends on the size of the
protein we want to visualize).

Loading buffer composition: Reducing buffer (with β-mercaptoethanol) allows to obtain monomeric
structures while non-reducing buffer helps to keep the quaternary structure of the protein.

Cow milk protein profile is highly complex. We will analyze the protein profile of centrifuged raw cow milk
+/- casein.

PRE-LAB QUESTIONS

A: Draw a schematic electrophoresis system detailing its working principle:

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B: Detail the main SDS-PAGE application:

C: Draw on the gel schematic the bands you would expect for the following samples treated in non-reducing
and reducing conditions:

1. a protein of 83kDa without quaternary structure. 5. a protein of 124 kDa formed by 2 equal subunits.
2. a protein with quaternary structure formed by 3 6. a mix of 3 proteins without quaternary structure of
subunits (30+62+77 KDa). 25, 410 and 500 kDa.
3. a protein of 3kDa without quaternary structure. 7. BSA protein
4. a protein with quaternary structure formed by 8. Gamma globulin
3subunits (50+50+88KDa).

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D: Analyze the differences between theses two prestained Protein Markers run under different SDS PAGE
conditions. What happens to each marker ? Why ?

E: Which are the main cow milk proteins ? Which protein profile can we expect to visualize with reducing
buffer SDS-PAGE with and without casein?

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MATERIAL AND REAGENTS

A: SDS-PAGE

A.1: Material

Electrophoresis power supply.

Electrophoresis equipment.

Figure 1 Components: A,
Electrophoresis module: Mini
Tank and Lid; B, Glass plates
and combs; C, Sample loading
guides; D, gel casting
components.

A.2: Reagents (already prepared)

Note: several reagents are toxic or harmful. It is required to avoid contact and inhalation and use gloves to
manipulate them.

PMW: Molecular weight marker (Bio-Rad All Blue) Storage at

-20ºC.

It’s a commercial mix of proteins of different molecular weight.

It will be used to estimate our protein molecular weight (MW).

Stacking gel: (CAUTION!: Neurotoxic)

Tris-HCl 0.5M pH 6.8 125 mM

SDS (10%) 0.1% (p/v)
Acrylamide 30% (Neurotoxic!) 4%
Distilled water -

Resolving gel: (CAUTION!: Neurotoxic)

Tris-HCl 1.5M pH 8.8 375 mM

SDS 10% (p/v) 0.1% (p/v)
Acrylamide 30% (Neurotoxic!) 10% (p/v)
Distilled water -

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APS (Ammonium PerSulphate) (10% p/v) (CAUTION!: harmful)

TEMED (liquid) (CAUTION!: harmful)

Electrophoresis buffer

Volume: 1 L Dissolvent: distilled water

REAGENT Concentration MW
Tris 25 mM 121.14
Glycine 192 mM 75.07
SDS 10% (p/v) 0.1 % (p/v) -

pH adjustment not required (aprox. 8.6).

Reducing sample buffer 2X

REAGENT Amount
Tris.HCL 2M pH 6.8 2 mL
Glycerol 8 mL
SDS 0.8 g
Bromophenol Blue 2.5 mg
b-Mercaptoethanol 100mM
Water 10ml
Total Volume 20 mL

Non-reducing sample buffer 2X

REAGENT Amount
Tris.HCL 2M pH 6.8 2 mL
Glycerol 8 mL
SDS 0.8 g
Bromophenol Blue 2.5 mg
Water 10ml
Total Volume 20 mL

B: Coomassie blue staining.

B.1: Material

Two glass Petri plates Plastic Pasteur pipette VersaDoc™ imaging system

B.2: Reagents (already prepared)

Note: Staining solution contains methanol (avoid methanol inhalation). Colorants can be carcinogens (avoid
skin contact).

Coomassie Blue staining solution (CAUTION!: Toxic)

Coomassie blue brilliant R-250 0.1 % (p/v) in Methanol 40% (v/v) and acetic acid 10%.

Destaining solution

Ethanol 40% (v/v) Acetic acid 3% (v/v) in distilled water

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C: Milk casein removal

Citric Acid 0.1M pH 4.6

D: Samples

Protein samples 1,2 and 3. 1 ml of raw milk (1 Eppendorf tube)

PROCEDURE

1. Plates assembly:

Since we are going to run a biphasic gel, each subgroup will have to polymerize two gels in one single glass
assembly; first the resolving gel will be prepared, then the stacking gel will be polymerized on top of the
resolving gel.

1.1. Carefully clean all the necessary components, specially the glasses. You need a glass holder, one
polymerization holder, one short glass and one outer glass (which includes the separators).

1.2. Joint the two glasses one in front the other with the separators in between (fig. 3a).

1.3. Open the clamps of the glass holder and insert in them the two glasses (fig. 3b). Take into account that
the holder must be the up side up.

1.4. Place the glass holder and the glasses on a flat surface so that the two glasses stand on the flat surface
before you tighten the clamps (fig. 3c).

1.5. Assemble the glasses and the glass holder in the polymerization holder (fig. 3d). Draw a black mark at 5
cm from the bottom with the waterproof pen. This will be the limit between the two gels.

1.6. Check the watertightness of the space between the glasses by pooring destilled water in. Once you are
sure of the watertightnes, without separating the holders, empty the water by decantation and wipe the water
drops with a filter paper.

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2. Resolving gel preparation (under hood):

2.1. Each subgroup will be provided with a 5 ml solution of 10 % gel in a 15 ml Falcon tube.

2.2. Calculate the amount of each reagent in the provided solution:

Required volume: 5 mL Dissolvent: distilled water

REAGENT Concentration Required amount:
Tris-HCl 1.5M pH 8.8 375 mM
SDS 10% (p/v) 0.1% (p/v)
Acrylamide 30% (Neurotoxic!) 10% (p/v)
Distilled water -

Calculations:

2.3. Add polymerization catalyzers in order to polymerize gel (mix well). Ensure electrophoresis casting
system is correctly assembled and hermetic before adding catalyzers!!

Polymerization catalyzers required for 5 ml of resolving gel

REAGENT Required amount
APS (Ammonium persulphate) 10 % 25 µL
TEMED 5 µL

2.4. IMMEDIATELY: add gel between glass plates with a plastic Pasteur pipet. Place pipet extreme over
short glass plate and load the gel until you reach 1-2 mm over the 5 com line. Do not discard the rest of the
polymerization gel (allow it to polymerize inside the 15 ml Falcon tube).

2.5. Add slowly 500 microlitres of Isopropanol over the added gel with a P1000 (It avoids the contact of the
solution with the oxygen in the air which would inhibit the polymerization reaction.)

2.6. Allow gel polymerization (20-30 minutes). Check spare gel polymerization in the Falcon tube.

2.7. Decant isopropanol when gel is polymerized. Remove it completely using absorbent paper.

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3. Stacking gel preparation (under hood):

3.1. Each subgroup will be provided with a 2,5 ml solution of 4 % gel in a 15 ml Falcon tube.

3.2. Calculate the amount of each reagent in the provided solution:

Required volume: 2,5 mL Dissolvent: distilled water

REAGENT Concentration Required amount:

Tris-HCl 0.5M pH 6.8 125 mM
SDS (10%) 0.1% (p/v)
Acrylamide 30% (Neurotoxic!) 4%
Distilled water -

Calculations:

3.3. Add polymerization catalyzers in order to polymerize gel (mix well). Ensure all is correct before adding
catalyzers!!

Polymerization catalyzers required for 2,5 ml of stacking gel

REAGENT Required amount

APS (Ammonium persulphate) 10 % 12,5 µL
TEMED 2,5 µL

3.4. IMMEDIATELY: complete spacers filling with stacking gel using a plastic Pasteur pipet. Place pipet
extreme over short glass plate and load the gel until you reach the top. Do not discard the rest of the
polymerization gel (allow it to polymerize inside the 15 ml Falcon tube).

3.5. Introduce a clean comb. IMPORTNANT: comb should enter smoothly (if it does not enter smoothly
ensure is not inversely placed: flat com side should face large glass). Avoid bubble formation (specially at
the bottom of the wells).

3.6. Wait until polymerization is ready (20-30 min.) Now, the gel is ready to be used.

IMPORTANT NOTES:

Glass should not be removed from frames and combs should not removed from gel until use.

Do not discard unpolymerized acrylamide. Check with teacher how to proceed.

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4. Samples preparation:

4.1 Tubes with BSA and gamma globulin:

For each sample we will prepare 100 μl of electrophoresis sample mixture (50 μl of sample plus 50 μl of
loading buffer (reducing or non-reducing loading buffer). We will obtain 6 samples.

4.1.1. Before pipetting label 6 Eppendorf tubes: 1, 1R, 2, 2R, 3, 3R.

4.1.2. Add to each of the Eppendorf tubes 50 μl of the corresponding sample (1, 2 or 3).

4.1.3. Add to the Eppendorf tubes of non-reducing conditions, 50 μl of the non-reducing loading buffer.

4.1.4. In the hood, add to the Eppendorf tubes of reducing conditions (R) 50 μl of the reducing loading
buffer.

4.1.5. Make a hole in the lid of each tube and heat the tube at 95ºC for 3 minutes.

4.1.6. Keep samples at 4ºC until gel loading.

4.2 Milk samples:

We will prepare a single dilution (1/10) of a centrifuged raw cow milk sample +/- casein (we will run 2
samples). For each sample we will prepare 100 μl of electrophoresis sample mixture (50 μl of sample plus 50
μl of reducing loading buffer).

4.2.1 Centrifuge 1 ml of raw milk Eppendorf tube the tubes 5 minutes at 14.500 rpm. Discard the pellet and
the creamy layer.

4.2.2 Repeat the procedure to ensure complete sample clearance.

4.2.3 Place 200 microlitres of the clear sample in a new labeled Eppendorf with 20 microlitres of 0.1M Citric
Acid (casein removal sample). Keep the rest of the clear sample to further analyze (initial milk sample).

4.2.4. Vortex casein removal sample sample 5 minutes at 14.500 rpm (a precipitate should appear).

4.2.5. Centrifugate casein removal sample 5 minutes at 14.500 rpm. Keep the supranedant in new labeled
Eppendorf tube (milk without casein sample) but do not discard the pellet (casein sample).

4.2.6. Label 3 tubes (Milk, Milk-C and C). Add to these 3 new labeled Eppendorf tubes 5 μl of the
corresponding sample (initial milk sample, milk without casein sample and casein sample) and 45 μl of
water. Note: in the casein sample add a small amount of the pellet.

4.2.7. Add to the Eppendorf tubes 50 μl of reducing loading buffer.

4.2.8. Make a hole in the lid of each tube and heat the tube at 95ºC for 3 minutes.

4.2.9. Keep samples at 4ºC until gel loading.

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5. Cuvette assembly and samples loading.

Remove gel comb cautiously to avoid wells damage.

Open electrophoresis module clamps (fig 4a).

Place both gels inside electrophoresis module short glass plates

facing inside (fig 4b). Ensure glass touch inferior side (fig 4a.
circles)

Close module clamps (fig. 4c). We have an interior chamber in

contact with the cathode (-) and the superior part of the gel. The
inferior part of the gel is in contact with the exterior anode (+)
(fig 4d).

Place electrophoresis module inside electrophoresis cuvette (fig

5). Ensure teacher has reviewed the assembly.

Fill interior chamber with electrophoresis buffer up to 2 mm of

the large glasses.

Fill exterior chamber with electrophoresis buffer up to 1 cm of

the large glasses .

Remove all water drops over glasses to avoid possible contact

between chambers avoiding pass through the gel.

Place loading guide between gels.

Add samples using a withe tips pipette (change each sample).

Add samples inside the wells following the guide.

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Place cuvette close to electrophoresis power supply. Chose a place where you can see electrophoresis
evolution without moving the cuvette.

Load:

• 7μl of molecular weight marker in the corresponding wells

• 10 μl of sample in the corresponding wells.

Load samples smoothly.

Make a diagram to record the position of the different samples in the gel describing the composition
of each sample.

Remove loading guides before placing the cuvette cover. Ensure cable colors combine with electrode color
(black + black and red + red)

Place the lid of the cuvette and connect to the power source.

6. Electrophoresis.

It is required a 35 mA fixed intensity to run each gel (70 mA if you are running 2 gels simultaneously). It is
also possible to run 2 cuvettes with 2 gels simultaneously (fixed 140 mA is required).

Switch on electrophoresis power supply with the following conditions :

Voltage maximum (300 V)

Intensity: 35 mA x gel

Connect cables between electrophoresis power supply and cuvette (CAUTION: cables connections should be
always of the same color).

Start electrophoresis. Bubbles should be observed in the electrode placed inside the interior chamber
(cathode). Intensity should be always constant and voltage can vary. Nevertheless, if voltage is very high it
could mean buffer in insufficient in the interior chamber and low voltage could mean a contact between
chambers avoiding the gel.

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It is required to check cuvette and electrophoresis power supply during electrophoresis. Check interior
cuvette level (refill if required but ensure to halt power supply before opening the cuvette cover).

Electrophoresis can be followed using pre-stained molecular markers (in 10-15 minutes samples are expected
to reach running buffer). Bromophenol blue runs faster than most proteins (MW 670) and a change of sample
behavior is clearly observed when it leaves stacking buffer and runs through running buffer (the density of
the acrylamide is significantly different).

Stop electrophoresis when bromophenol blue reaches approximately 5 mm to the inferior end of the gel
(inferior part of glass plates).

7. Gels extraction.

We will stain both gels with Coomassie blue. Prepare 2 Petri plates with approximately 25 mL of Coomassie
blue solution.

Remove electrophoresis module from the cuvette. Empty electrophoresis cautiously inside the cuvette.
Unassemble clamps and remove plates containing the gels.

Separate glass plates using the plastic separator (ask the teacher). Gel will be stuck to one of both glass
plates.

Remove the gel from the glass plate with extreme caution as it is very fragile (ask the teacher).

Place each gel inside the Coomassie solution Petri plates.

Allow gels to stain (go to section 3). Clean glass plates immediately with tab water and rinse with distilled
water. Allow them to dry.

Recover electrophoresis buffer. Carry out buffer recovery in a sink using a funnel.

Clean all components (except cuvette green cover) with tap water and rinse with distilled water.

8. Coomassie blue staining and destaining.

Coomassie staining allows a blue staining of all proteins present in the gel.

Cover Petri plate with the gel and 25 ml of Coomassie blue staining solution and allow staining for 15
minutes.

Return staining solution to its bottle using a plastic Pasteur pipette (caution). The gel should be uniformly
deep blue.

First destaining: place approximately 60 ml of destainning solution inside the Petri plate containing each gel.
Keep for 20 minutes at room temperature over the rotary shaker.

Second destaining: place approximately 60 ml of a new destainning solution. Destain until gel blue
background disappears. Recover destainning solution as indicated previously.

Take a gel image picture.

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RESULTS AND DISCUSSION

A: Using the electrophoresis gel you have performed in the laboratory, represent on the logaritmic paper an
standard curve (MW vs electrophoretic mobility) and interpolate the electrophoretic mobility of your
samples (all the bands) to find their MW.

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B: Compare you results with the other subgroups:

Obtained bands size:

Subgroup 1 Subgroup 2 Subgroup 3 Subgroup 4 Subgroup 5 Subgroup 6
S 1 reducing

S 1 non
reducing
S 2 reducing

S 2 non
reducing
S 3 reducing

S 3 non
reducing
Milk

Milk - Casein

Casein

C: Which protein/s contain the Samples 1, 2 and 3 ? Explain how did you find it.

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D: Can you identify proteins from the two milk samples (Milk and Milk - Casein) ?

E: Why sample pre-treatment is critical in SDS-PAGE ?

SAFETY CONSIDERATIONS

Follow laboratory safety considerations.

Wear gloves and safety glasses when manipulating materials and reagents.

Specific care:

Several reagents are toxic or harmful. It is required to avoid contact and inhalation and use gloves to
manipulate them. Do not discard unpolymerized acrylamide. Check with teacher how to proceed.

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SESSION 3: Bacterial DNA purification and polymerase chain reaction (PCR)

OBJECTIVES

A) Primary raw milk sample preparation and bacterial DNA extraction.

B) Amplify bacterial DNA present in raw milk samples by PCR.

C) Visualize and interpret PCR amplified DNA by electrophoresis using two different amplification
protocols.

BACKGROUND

Raw cow milk: It sometimes contains a wide range of different bacteria. High bacterial loads are often
related to undesired situations like Mastitis. PCR can be used to identify an quantify the presence of bacteria
in raw cow milk.

DNA purification is key to correctly perform techniques based on DNA. For instance, the way raw milk
samples are processed can strongly influence bacterial DNA detection by PCR.

PCR is a widely used set of different techniques to amplify DNA and RNA. These techniques are based on
amplifying a certain sequence by a consecutive cycles of Denaturation, Annealing and Extension/elongation.

Agarose electrophoresis is widely used technique to visualize DNA and RNA. This technique allows to
separate DNA and RNA molecules based on their size.

PRE-LAB QUESTIONS

A: Different bacteria species are often present in raw cow milk (specially in cows with Mastitis). Which
PCR techniques do you recommended to detect and quantify different bacteria in cow milk ? Which are the
advantages and limitation of each technique ?

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B: Explain Proteinase K role in DNA purification

C: Draw a PCR schematic procedure to amplify bacterial DNA using PCR (Denaturation, annealing,
extension/elongation)

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D: Explain this graphic:

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E: Draw a Agarose Electrophoresis schematic procedure to visualize DNA.

F: What can we amplify with the following primers ? Which product size can we expect to obtain with
331F and 797R primers ?

(Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set.
Mangala A Nadkarni et al. 01 January 2002 https://doi.org/10.1099/00221287-148-1-257 )

331F TCC TAC GGG AGG CAG CAG T

797R GGA CTA CCA GGG TAT CTA ATC CTG TT

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MATERIAL AND REAGENTS

PCR sample preparation:

- 2 Raw milk samples (1 ml each in eppendorf tube)

- PBS + 0,1 % Tween

- 500 mM EDTA

- Lysis buffer A (10 mM Tris, 1 mM EDTA pH8, 0,5% Tween and 200 µg/mL proteinase K)

PCR reaction:

- BIOTED PCR gen 16S ARNr bacteriano Ref. PCR16S

- DNA free water (negative control)

Agarose electrophoresis:

- 1 X TAE (40 mM Tris-base, 20 mM Acetic acid, 1 mM EDTA)

- Agarose (0,75 g/gel)

- SYBRTM Safe DNA (10000x, Invitrogen) (5µl / gel)

- PCR 100bp Low Ladder (MW) with loading buffer

Equipments:

- Vortex

- Eppendorf tubes centrifuge

- Thermal block

- PCR tubes centrifuge

- Thermocycler

- Agarose gel electrophoresis equipment (50 ml gel + 6 teeth combs)

- Microwave oven

- UV-transilluminator

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PROCEDURE

1. PCR sample preparation:

A) Initial sample cleaning.

- Thaw 2 raw milk samples (1 ml in each eppendorf tube).

Milk sample 1 name: Milk sample 2 name:

- Add the required 500 mM EDTA volume to each tube to obtain a final concentration of 40 mM EDTA in
each tube.

EDTA volume:

- Vigorous mix each tube with vortex during 10 minutes (tubes can be optionally fixed to vortex with tape).

- Centrifuge straightaway the tubes 5 minutes at 14.500 rpm.

- Remove the supranedant (specially care should be taken to remove the lipid layer).

- Resuspend the pellet with 1 ml PBS + 0,1 % Tween.

- Centrifuge the tubes 5 minutes at 14.500 rpm.

- Remove the supranedant again.

B) Proteinase K lysis.

- Add 500 microlitres of lysis buffer A (10 mM Tris, 1 mM EDTA pH8, 0,5% Tween, and 200 µg/mL
proteinase K) in each tube with the pellet.

Note: add proteinase K at 6 mg/ml just before buffer addition.

Lysis buffer A volume without proteinase K:

Proteinase K (6 mg/ml) volume:

- Vigorous mix with vortex during 1 minute and incubated at 55°C for 1 h.

- Heat at 95°C for 10 min to inactivate the proteinase K.

- Centrifuge 5 minutes at 14.500 rpm to remove pellet.

- Keep supernatant in a new Eppendorf tube (label it as Proteinase K milk sample + name and date).

Proteinase K milk sample 1 name: Proteinase K milk sample 2 name:

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2. PCR reaction:

Two PCR programs will be performed with each sample (25 and 35 cycles). Each subgroup will use one of
the two programs. Negative control will be prepared by the teacher.

- Mix reagents (see details in the following table).

REAGENT S1 S2 Positive control (P)

PCR Mix 22. 5 μl 22. 5 μl 22. 5 μl

BIOTED PCR gen 16S ARNr
bacteriano
Samples and controls 2.5 μl Proteinase K 2.5 μl Proteinase K 2.5 μl positive control
milk sample 1 milk sample 2
Total Volume 25 μl 25 μl 25 μl

- Mix well all reagents (polymerase red colour helps to achieve a good mixing)

- Short centrifugation using a PCR tubes centrifuge (5 seconds at 2000 rpm)

- Place tubes at the thermocycle and use assigned BITPCR program.

BITPCR program:

BITPCR program 1 has the following profile (total time program is approximately 2 1/4 hours):

STEP Temperature Time

1 95ºC 10 minutes
2 95ºC 60 seconds
25 cycles
3 55ºC 60 seconds
4 72ºC 60 seconds
5 72ºC 10 minutes
6 4ºC Infinite

BITPCR program 2 has the following profile (total time program is approximately 2 and 1/2 hours):

STEP Temperature Time

1 95ºC 10 minutes
2 95ºC 60 seconds
35 cycles
3 55ºC 60 seconds
4 72ºC 60 seconds
5 72ºC 10 minutes
6 4ºC Infinite

3. Agarose electrophoresis: (prepare 1 agarose gel / subgroup)

- Close cuvette open sides with tape in order to avoid agarose leak.

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- Prepare a 50 ml 1,5 % g of agarose in TAE X 1 using TAE X 10

REAGENT UNITS REQUIRED AMOUNT

Agarose Weigh
TAE X1 Volume
Water Volume
SYBRTM Safe DNA X 10.000 Volume

- Mix reagents and heat until boiling in a Microwave oven.

- Ensure agarose is correctly dissolved. Add with SYBRTM Safe DNA X 1 when solution temperature
decreases to approximately 50ºC.

- Remove to ensure complete mixing and pour all the volume carefully in the electrophoresis cuvette.

- Place a 6 wells comb.

- Allow to solidify (place in the fridge if required).

- Place agarose gels in a cuvette. Fill the electrophoresis cuvette with TAE X1.

- Remove gels combs carefully.

- Load agarose gel (1 subgroups / gel). Add 20 µl of each sample to the corresponding lane except MW and
Negative control (volume defined by the teacher).

Lane Sample
1 Negative Control (W) Provided volume
2 Positive Control (P)
3 Proteinase K milk sample 1 (S1)
4 Proteinase K milk sample 2 (S2)
5 PCR 100bp Low Ladder (MW) Provided volume

- Run gel at 100 V for about 25 minutes.

- Visualize results in a UV-transilluminator. Take a picture.

- Return TAE 1X to its original bottle for further use.

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RESULTS AND DISCUSSION

A: Draw the electrophoresis bands and assign a qualitative value (from 0 -no band- to 5 -maxim intensity) to
each lane without any band (0) or just a single well defined band (1 to 5).

B: Plot all subgroups results (only fill performed assays)

Sample SG1 SG2 SG3 SG4 SG5 SG6

BITPCR 1 Negative Control (W)
BITPCR 1 Positive Control (P)
BITPCR 1 Proteinase K milk sample 1
BITPCR 1 Proteinase K milk sample 2

BITPCR 2 Proteinase K milk sample 2

BITPCR 2 Proteinase K milk sample 1
BITPCR 2 Positive Control (P)
BITPCR 2 Negative Control (W)

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C: Discuss the obtained results. Consider your data as well as the data obtained with the other subgroups.

D: Which are the key parameters you should optimize to set up a PCR to detect bacteria in raw cow milk ?

SAFETY CONSIDERATIONS

Follow laboratory safety considerations.

Wear gloves and safety glasses when manipulating materials and reagents. Take special care with PCR
contaminations as small amount of templates can be amplified.

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