JOMO KENYATTA UNIVERSITY OF AGRICULTURE AND

TECHNOLOGY

BSC. MEDICAL LABORATORY SCIENCES

COURSE TITLE: MEDICAL BIOTECHNOLOGY

COURSE CODE: MLS 2407

TOPIC: PRACTICAL REPORT

LECTURER:

PROF. JULIETTE ONGUS

MEMBERS
1. MERCY CHEPKIRUI HSB231-0572/2023
2. SHEILAG TUNE HSB231-0421/2023
3. SAMUEL OKOTH HSB231-0422/2023
4. JEPKOSGEI CAROLINE HSB231-0461/2023
BUFFER PREPARATION: PHOSPHATE BUFFER SALINE

INTRODUCTION

Phosphate buffered saline (PBS) is a common buffer solution used in biological research. It's a
salt solution containing sodium chloride, sodium phosphate, and potassium phosphate. PBS is
often used in cell culture, biochemistry, and molecular biology applications

OBJECTIVE

To study how to prepare phosphate buffer saline.

Requirements

Sodium chloride

Potassium chloride

Beaker

Distilled water

Ph meter

hydrochloride acid

potassium dihydrogen phosphate

disodium hydrogen phosphate

spatula

analytical

balance

stirring bar
PROCEDURE

Calculations

1. 500mls of 10X PBS buffer was prepared

SALT FORMU MOLECULAR CONCENTRATI CONCENTARTION X

LA MASS(gms/mo ONS (mmol/l) S(g/l) 10
les)
Sodium NaCl 58.5 140 8.12 81.
chloride 2
Potassiu KCl 74.5 2.7 0.20 2
m
chloride
Disodiu NaH2PO4 120 10 1.2 12
m
hydroge
n
phospha
te
Potassiu K H2PO4 136 1.8 6.24 62.
m 4
dihyrog
en
phospat
e
PH -
2. The component was dissolved in 400mls distilled water using a magnetic stir
3. The PH was adjusted to 7.4 with concentrated HCL.
4. The volume was adjusted to 500mls with addition of distilled water.
5. The solution was autoclaved for sterility.
6. The PBS solution was transferred to a clean labelled storage bottle.
Result

Clear colorless solution was attained which had a PH of 7.4

DISCUSSION
PBS maintains cell viability and prevent osmotic shock during procedures.

CONCLUSION
The preparation of PBS was successfully completed. with a final PH of 7.4, suitable for
biological application.
References
Lab manuals
Lecture notes

PRACTICAL 2
PIPETTING TECHNIQUES
INTRODUCTION
Pipetting is a fundamental laboratory technique used to accurately transfer small volumes of
liquids. Proper pipetting technique is crucial for obtaining reliable and reproducible results in
various scientific experiments.
Micropipettes are used for handling volumes ranging from 0.1 µL to 1000 µL, whereas
serological pipettes handle larger volumes (1 mL to 25 mL).
Types of Pipetting Techniques
1. Forward Pipetting: Used for standard aqueous solutions.
2. Reverse Pipetting: Ideal for viscous or foaming liquids.
 Forward Pipetting: The most common technique, where the desired volume is aspirated and
then dispensed.
 Reverse Pipetting: Used for viscous liquids or those that tend to foam. In this technique, the
plunger is pressed to the second stop before immersing the tip in the liquid. This creates an air
cushion that prevents the liquid from entering the pipette shaft.

OBJECTIVE

To learn how to perform reverse and forward pipetting techniques

 REQUIREMENTS

Micropipettes (P10, P100, P1000)

Pipette tips
Pipette aid
Distilled water
Beakers
Analytical balance
Weigh boats
Safety box (waste container)

PROCEDURE

PIPETTE MEASUREMENTS

FORWARD PIPETTING TECHNIQUE REVERSE PIPETTING TECHNIQUE

(250ul pipetted) (250ul pipetted)
1 0.244 0.2517
2 0.2443 0.2596
3 0.344 0.2480
4 0.243 0.2476
5 0.2436 0.2485
6 0.2433 0.2465
7 0.2542 0.2480
8 0.2428 0.2469
9 0.2442 0.2555
10 0.2441 0.2470
Mean 2.4475/10 = 0.24775 2.4993/10 = 0.24993
Forward Pipetting Technique:

1. 250ul volume was set on the pipette and the appropriate pipette tip was fixed onto the pipette.

2. The pipette plunger was pressed to the first stop (to expel air) and the tip was immersed into
the distilled water and slowly released the plunger to draw the liquid into the tip. It was ensured
no air bubbles were present.

3. The plunger was pressed to the first stop to release the distilled water, and then pressed the
plunger fully to the second stop to expel any remaining distilled water into the weigh boat and
was weighed using an analytical balance and the reading was recorded.

4. The procedure was repeated 10 times.

5. The pipette tip was removed and dispose of it in a waste container. Ensure the pipette is
cleaned and maintained.

Reverse Pipetting Technique:

1. 250ul volume was set on the pipette and the appropriate pipette tip was fixed onto the pipette.

2. The pipette plunger was pressed to the last stop (to expel air) and the tip was immersed into
the distilled water and slowly released the plunger to draw the liquid into the tip. It was ensured
no air bubbles were present.

3. The plunger was pressed to the first stop to release the distilled water to the weigh boat and
was weighed using an analytical balance and the reading was recorded. The excess distilled
water on the pipette tip was discarded.

4. The procedure was repeated 10 times.

5. The pipette tip was removed and dispose of it in a waste container. Ensure the pipette is
cleaned and maintained.

RESULTS
There was consistency in pipetting
DISCUSSION
Forward pipetting is generally preferred for most aqueous solutions and when high accuracy is
critical for small volumes while reverse pipetting Viscous liquids (e.g., glycerol, oils) Foaming
liquids (e.g., detergents, protein solutions)

CONCLUSION
forward and reverse pipetting techniques are key skills in medical biotechnology. Accuracy in
both techniques ensures accurate and reliable results in experiments, which is vital for the
success of medical biotechnology applications, including diagnostics, research, and therapeutic
development

REFERENCE
Lab manuals.
Lecture notes.
 PRACTICAL 2

DNA EXTRACTION FROM BLOOD SAMPLE

INTRODUCTION

DNA extraction from blood is a common technique used in various fields, including forensic
science, medical diagnostics, and research. The process involves isolating DNA from blood cells,
which can then be used for further analysis, such as PCR, sequencing, or genotyping.

PRINCIPLE

The DNA extraction process involves breaking open the cells (lysis) to release the DNA into
solution. This is typically done using a lysis buffer that contains detergents like SDS to disrupt
cell membranes and proteins like Proteinase K to degrade proteins. The DNA is then precipitated
using alcohol (isopropanol or ethanol) and purified by washing with alcohol to remove
contaminants. The extracted DNA is quantified using a UV-Vis spectrophotometer by measuring
the absorbance at 260 nm, which corresponds to DNA, and at 280 nm, which corresponds to
proteins. The ratio of absorbance at 260 nm to 280 nm (A260/A280) is used to assess the purity
of the DNA.

OBJECTIVE

To learn how to perform DNA extraction from blood using a standard DNA extraction method.

REQUIREMENTS:
Reagents:
Blood sample (whole blood)
Lysis buffer (SDS)
Proteinase K
Isopropanol
Ethanol (70%)
Equipment
Centrifuge
Microcentrifuge tubes
UV-Vi’s spectrophotometer
Incubator
Vortex mixer

Consumables:
PPEs (Gloves, lab coat, safety
glasses)
Pipettes
Disposable sterile pipette tips
Microcentrifuge tubes (1.5 mL)
Red blood cell lysis buffer
WBC cell lysis buffer
Saturated sodium chloride solution
Absolute ethanol
TE buffer
PROCEDURE

1. 1 mL of whole blood was transferred to a 15 mL centrifuge tube.

2. 10 mL of RBC lysis buffer was added and mix gently by inverting the tube.

3. The mixture was incubated at room temperature for 10 minutes and inverting occasionally.

4. The mixture was centrifuged at 1,500 rpm for 10 minutes.

5. The supernatant was discarded leaving a white pellet of WBCs.

6. The white blood cells pellet was resuspended in a 1Ml of WBC lysis buffer

7. 20ul of proteinase K was added to the mixture and incubated at 56oC for an hour to digest
proteins.

8. 300ul of saturated sodium chloride solutions was added to the lysate. And the mixture was
vortexed vigorously for 15 seconds to precipitate proteins.

9. The mixture was centrifuged at 12000 RPM for 10 mins.

10. The clear supernatant was transferred to a new tube.

11. 2 volumes of cold absolute ethanol was added to the supernatant and mixed by inverting.
Formation of a white thread-like DNA precipitate was observed.

12. The mixture was centrifuged at 12000RPM for 5 minutes to collect the DNA.

13. The DNA pellets was washed with 500ul of 70% ethanol. The pellet was airdried for 10
minutes.

14. The DNA was resuspended in a 100ul of TE buffer.

DNA QUANTIFICATION AND PURITY ASSESSMENT

1. DNA concentration was measured using spectrophotometer absorbance at 260-280nm.

RESULTS
The absorbance at 260-280nm was 1.85 indicated good quality DNA.
DISCUSSION
The salting-out method is efficient for isolating DNA from WBCs, producing high-quality DNA
with minimal protein contamination.
The use of saturated NaCl effectively precipitated proteins without requiring hazardous organic
solvents like phenol.
Cold ethanol ensured effective DNA precipitation.

CONCLUSION
High-quality genomic DNA was successfully extracted from white blood cells using the salting-
out method. The procedure is simple, cost-effective, and suitable for routine DNA isolation for
molecular biology applications.

REFERENCE
Lab manuals.
Lecture notes.