Cell Biology Lab

Lab Tech Manual

BIOL 211

Prepared by: R. Stefan Rusyniak

COLLEGE OF ARTS AND SCIENCES

Department of Biological and Environmental Sciences
Qatar University
Lab / Week: Week 1 – No labs
Materials needed:
Part 1:
Part 2:
Part 3:
Lab / Week: Week 2 – Intro to Lab and Lab Safety, and Cells
Materials needed:
Part 1: nothing
Part 2: nothing
Notes:
Lab / Week: Week 3 – Reagent Calculations, Preparation of Stock Solutions
Materials needed:
Part 1: Nothing
Part 2: Per Group
• 250ml Beakers (3)
• 100ml Graduated Cylinder (1)
• 50ml Falcon Tubes (4) (autoclavable)
• 100ml bottles (2) (for autoclaving)
• Stir plate
• Small (~2-3cm long) magnetic stirrers (3)
• pH meter, pH standards, waste beaker and squirt bottle
• Kimwipes
• 1M HCl, 0.1M HCl, 1M NaOH, 0.1M NaOH
• Droppers for acid and base solutions
• Parafilm

Reagents
• Sucrose mol wt - 342.3 g/mol
• NaCl mol wt - 58.44 g/mol
• KCl mol wt - 74.55 g/mol
• Na2HPO4 · 7H2O mol wt - 268.1 g/mol
• NaH2PO4 · H2O mol wt - 138.0 g/mol
• MgCl2 mol wt - 203.3 g/mol
• Succinate mol wt – 118.09 g/mol

For the class

• Masking tape for labeling
• Autoclave indicator tape
• Sharpies
• Scale (x 2) and plastic weigh boats
• Clean steel spatulas or plastic spoons (not the glass ones)

Notes:

Please make sure that the caps are not too tight on the bottles and falcon tubes before
autoclaving

Please autoclave the solutions and return them to the lab. Please be sure to close the caps
once the solutions have cooled down and try to keep each groups solutions easy to identify.
Lab / Week: Week 4 – Volumetrics & Scientific Communication
Materials needed:
Part 1: Solutions prepared in the previous week

Per Group
• test tube rack for Falcon tubes (each group in each section will need their own rack)
• 50ml Falcon tubes (5)
• 1ml, 2ml and 5ml pipettes (4-5)
• pipette aid (2)
• 50ml graduated cylinder
• parafilm

Sharpies

Part 2: per group

• 100ml 10% sucrose solution (dyed any color)

• 100ml each of distilled water and water dyed blue
• small beakers or flasks (4)
• 1.5ml micro-centrifuge tubes (12 per group member)
• Micro-centrifuge tube rack
• set of 3 micropipettes (P20, P200, P1000)
• box of 20-200µl tips
• box of 100 – 1000µl tips
• wash bottle with 70% ethanol

micro-centrifuge
Part 3:
Notes:

Students will be preparing solutions for use in Week 7 and 8. Please store them until then.
Lab / Week: Week 5 – Slide Preparation, Kohler Illumination and Microscopy
Materials needed:
Part 1: Compound microscopes – 1 per student
Stained Slides

Part 2:
Per Student:
• 2 slides with mounted sections

Two work stations with staining series:

• 100% Xylene (2)
• Ethanol series (100%, 95%, 90%, 80%, 70%, 50%)
• Hematoxylin
• Water (1)
• Ethanol series (50%, 70%, 80%, 90%)
• Eosin
• Water(1)
• Ethanol Series (95%, 100%, 100%)
• 100% Xylene (2)
• Canada Balsam – 1 bottle per work station with a small stick for application
• Box of Kimwipes (small)
• Clean coverslips

1 Slide warmer
Notes:
Lab / Week: Week 6 – Cell Counting and Viability
Materials needed:
Part 1: Compound microscope for each students
Counting chamber / Haemacytometer
70% ethanol squirt bottles and kimwipes
Bottle of water
Eppi tubes
Micropipettors (P20 and P200)
Trypan Blue stain

Trypsinized cell culture (provided by TA)

Part 2:
Notes:
Lab / Week: Week 7 – Cellular Fractionation
Materials needed:
Part 1: Solutions from Week 4

Per Group
• Syringe with a 26- or 28-gauge needle
• 2 centrifuge/falcon tubes (15ml)
• P1000 Micropipettor and tips
• Test tube rack
• Eppendorf tubes (epi tubes)
• Ice bucket with ice

Refrigerated Microfuge from C118

Clinical Centrifuge

Notes:
Lab / Week: Week 8 – Qualitative and Quantitative Determination of Sample Contents
Materials needed:
Part 1: Per Group:
• Cuvettes
• Spectrophotometer
• 20ml of Bradford Assay Buffer
• 1.5ml of 1M NaOH
• Ice bucket with ice for samples
• Eppendorf tubes and test tubes
• Micropipettors (P200 and P1000)
• 5ml PBS

Part 2: Compound microscope for each group

Clean slides and coverslips
Hematoxylin stain (~1ml in an eppi tube)
70% ethanol squirt bottles and kimwipes
Bottle of water
Eppi tubes
Micropipettors (P20 and P200)
Trypan Blue stain
Notes:
Lab / Week: Week 9 – Midterm
Materials needed:
Part 1: Nothing
Part 2:
Notes:
Lab / Week: Week 10 – Marker Enzyme Assay
Materials needed:
Part 1: Per Group
 ice in an ice bucket
 14 test tubes
 test tube rack
 spectrophotometry cuvettes
 spectrophotometer
 Micropipettors (P200 and P1000)
 Parafilm
 Eppendorf tubes
 4ml of 0.04M Sodium Azide or Potassium Cyanide
 2ml of 5mM Dichlorophenolindophenol (DCIP) – should be made fresh

For the class

 37C water bath
 Part 2:  100ul of 5X Denaturing Buffer (0.25M Tris pH 6.8, 10% SDS, 40% Glycerol,
20% β-mercaptoethanol, 0.01% Bromophenol Blue)
 ice in an ice bucket
 95C water bath or heating block
 eppi tubes
 Microfuge
 Micropipettors (P20 and P200) and tips
 SDS-PAGE Gel Apparatus
 SDS running buffer (enough for two gels)
 2 x 12% SDS-PAGE Gels
Notes:
To make 10 ml of 5x stock

Mix the following:

2.5 ml 1 M Tris-HCl pH 6.8
1.0 g SDS
0.5 ml 0.1% Bromophenol Blue
4 ml 100% glycerol
2 ml 14.3 M β-mercaptoethanol (100% stock)
Lab / Week: Week 11 – DNA Extraction
Materials needed:
Part 1: • 1ml Sample of induced cells
• 1ml Sample of uninduced cells (control)
• Eppendorf / Microfuge tubes
• Micropipettors (P200 & P1000) and tips
• 200ul of PBS (10mM PO 43- , 137mM NaCl, 2.7mM KCl)
• 1ml of Lysis Buffer (250mM NaCl, 1mM Tris, 10mM EDTA, 0.5% SDS, pH 7.5)
• RNase A
• Proteinase K
• 37C Water bath
• Microcentrifuge
Part 2: • 1ml of 8M Ammonium Acetate
• 3ml of 100% Ethanol
• 3ml of 70% Ethanol
• 200ul TE (10mM Tris, 1mM EDTA, pH 8)
Notes:
The Cell samples will be provided by Mr. Alaa in the Research Complex
Lab / Week: Week 12 – DNA Laddering
Materials needed:
Part 1: • 1.2% agarose gel
• 0.5X TBE Buffer for the gel tank
• Agarose gel tank
• Power Supply
• Loading dye
• P20 and P200 micropipettors
• Boxes of 20µl and 200µl tips
• Epi tubes
• Microcentrifuge
Part 2: • Sample of induced cells
• Sample of uninduced cells (control)
• PBS (10mM PO 43- , 137mM NaCl, 2.7mM KCl)
• Micropipettors (P200 & P1000) and tips
• Slides and coverslips
• NucBule fluorescent staining kit from Life Technologies
Notes:
Cells will be provided my Mr Alaa in the research complex