Lab Manual 1to5 W2020 Final-1 PDF

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BIOL301 – Cell and Molecular Laboratory LAB MANUAL

Winter 2020 Prof. Hugo Zheng (course coordinator) Prof. Rodrigo Reyes-Lamothe Anne-Marie Sdicu (lab coordinator)

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Background Reading A brief introduction has been included for each of the main techniques used in this course. Further information on basic techniques (PCR, gel electrophoresis, restriction digests, cloning) is in our recommended text – Lodish et al., Molecular Cell Biology, 8th Ed, which is the text you used for BIOL 200. We highly recommend you read the whole of Chapter 6 8th Ed. “Molecular Genetic Techniques”, as it covers many of the techniques used in Labs 1-5, as well as providing background for things discussed in the lectures. For Labs 6 to 10, we recommend you read Sections 4.2, 4.3 and 3.5 in Lodish 8th Ed. Detailed references for most of the experiments are in the Current Protocols laboratory manuals (Molecular Biology) available as eBooks at the McGill library. Other reference materials are on myCourses. Since a number of the basic techniques used in the labs are in this manual and in BIOL 200 and/or BIOL 202, the lectures for Labs 1-11 will not focus on their details. Instead, refer to the short backgrounds covered in the lab manual, as well as the pre-lab videos available through myCourses. Note: molecular biology techniques take time – there are often waiting times between steps within a procedure. This means a couple of things: (1) Come to the lab prepared – it is critical that you watch/read the required videos and notes ahead of the lab so that you know what you will be doing and be able to work efficiently in your pair and/or group. (2) There is no time to waste doing the experiments – when the first step is finished, you need to be ready to start the next step right away, or you will run out of time. Icons: there are three icons used in this manual to help you quickly identify different types of information. Safety: indicates important information about lab safety. Pay special attention to it for health risks and hazards you might encounter in the lab. Materials or equipment: indicates materials or equipment that you will be using in the lab. Pay attention to the information so that you know how to handle properly the materials or the equipment. Experimental protocol: indicates the start of the experimental protocol you will be following for a given lab. Pay close attention to the steps involved.

Links: website links for certain terms, techniques, and references have been included to provide you with further information that might be helpful in understanding the lab theory or the lab protocols (e.g., definitions for specific terminology).

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TABLE OF CONTENTS LABS Pages Lab 1. Introduction to Lab safety, Lab techniques & Molecular Biology

5-21

Lab 2. Introduction to PCR, Gel Electrophoresis & Data Analysis

22-32

Lab 3. Error-Prone PCR of mNeonGreen & Cloning Preparation

34-42

Lab 4. PCR purification & Cloning of mNeonGreen through Restriction Digest

43-52

Lab 5. Cloning Continued: Ligation & Transformation

53-58

APPENDICES Pages Lab 2. A2.1. Protocol for Basic PCR Reaction

60-61

A2.2. Analyzing Gel Images on a Laptop

62

A2.3. DNA Ladders Used in Labs 2-5

63

Lab 4. A4.1. Estimating DNA Quantity by Comparison to a Calibrated Ladder

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LABORATORY 1. INTRODUCTION TO LAB SAFETY, LAB TECHNIQUES & MOLECULAR BIOLOGY In this course, you will be carrying out a molecular biology research project to mutate a fluorescent protein and to characterize its function. Throughout the semester, you will learn to perform a variety of molecular techniques to help you address specific experimental questions related to your research project. Many of the techniques that you will be learning involve specialized equipment and/or chemicals that require careful use to avoid mistakes or injury. Today, you will be introduced to some of the equipment and chemicals you will encounter in the lab during the semester, and you will be instructed on their proper and safe use. You will then put your new lab skills to use with a short experiment that explores working with plasmid DNA and bacteria. LAB SAFETY Always wear lab-appropriate attire when working in the lab:     

Lab coat Goggles Gloves Shoes that cover your entire foot Tie long hair back

If you are unsure if something you are wearing is lab appropriate, ask one of your TAs or the lab coordinator. Note: always remove your gloves before touching any of the computers in the lab to prevent contaminating them with any of the samples or chemicals you are working with. If you are caught touching a computer with gloves one, you will get 0 credits for that lab and will be responsible for decontaminating the computer. At the beginning of Lab 1, we will explain some basics of working safely in the lab and how to get information about the potential hazard level of some of the chemicals used during the various lab exercises. Binders located in the lab contain the “Material Safety Data Sheets” (MSDS, https://www.ccohs.ca/oshanswers/legisl/msdss.html) arranged in alphabetical order. This information is also included in mylab (online). Instructions for how to access mylab are on the far wall in the lab near the printers. A Material Safety Data Sheet is a document that provides information on a controlled product. It outlines the product’s toxic effects, the protective measures for avoiding overexposure or chemical hazards, and the procedures to follow in an emergency. A controlled product is a hazardous material meeting the hazard criteria defined in the Controlled Products Regulations. The information provided on the MSDS completes the information found on the label of a controlled product. By law, the supplier is obligated to send the MSDS with the product sold. It must be on the premises in a location known by the users, and it must be easily and rapidly accessible to those who are likely to be in contact with the product. BIOL301

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INFORMATION DISCLOSED ON THE MATERIAL SAFETY DATA SHEET The Material Safety Data Sheet must contain nine categories of information. They are under the following headings or under equivalent headings:         

Product information Information on the preparation of the MSDS Hazardous ingredients Physical data Fire and explosion hazards Reactivity data Toxicological properties Preventive measures First aid measures

The internationally harmonized MSDS consists of 16 categories of information accepted in Canada. It contains the information required by WHMIS (Workplace Hazardous Materials Information System, https://tinyurl.com/ycfv656k) and the product is in accordance with the hazard criteria listed in the Controlled Products Regulations. For more information, you can go to the McGill Environmental Health & Safety and Biology Health and Safety. On chemical container labels, you will find pictograms that represent specific dangers: The pictogram represents...

The pictogram means...

And you should do this:

Class A Compressed Gas

 It is a gas kept under pressure.  Heat may cause the container to explode.  A drop or impact may cause the container to explode.

 Handle with care. Do not drop.  Keep away from heat or potential sources of ignition.  Store in a designated area.

Class B Flammable & Combustible Material

 The material is a potential fire hazard.  It may burn at relatively low temperature.  Sparks, flame or friction could ignite it.  May burst into flame spontaneously in air or release a flammable gas on contact with water.

 Keep the material away from heat sources and other combustible materials.  Never smoke when working with or near the material.  Store in a cool, fireproof area.

 The material is a fire or explosion risk near flammable or combustible material.  May burn skin or eyes in contact.

 Keep the material away from combustible materials and store in designated areas.  Keep the material away from sources of ignition.  Never smoke when working near the material.  Wear the proper protective equipment, including eye, face and hand protection and protective clothing.

 The material is a potentially fatal poisonous substance.  It may be fatal or cause permanent damage if inhaled, swallowed or absorbed through skin.  May burn skin or eyes on contact.

 Handle the material with extreme caution.  Avoid contact with the skin or eyes. Use proper protective clothing.  Avoid inhaling by working in well-ventilated areas and/or wearing respiratory equipment.  Wash and shower thoroughly after using.  Store in designated areas only.

Class C Oxidizing Material

Class D, Division 1 Poisonous & Infectious Material: Immediate & Serious Toxic Effects

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Class D, Division 2 Poisonous & Infectious Material: Other Toxic Effects

Class D, Division 3 Poisonous & Infectious Material: Biohazardous Infectious Material

Class E Corrosive Material

Class F Dangerously Reactive Material

 The material is poisonous but not immediately dangerous to health.  It may cause death or permanent damage if repeated exposure over time.  May be a skin irritant.  May be a sensitizer causing allergic reaction.  May cause cancer.  May cause birth defects or sterility.

 Avoid skin and eye contact by wearing all protective equipment necessary including eye, face and hand protection and protective clothing.  Avoid inhaling by working in wellventilated area and/or using respiratory equipment.  Store in designated areas.

 May cause a serious disease resulting in illness or death.

 Take every measure to avoid contamination.  Handle the material only when fully protected by the proper, designated equipment.  Handle the material in designated places only.

 Causes severe eye and skin irritation upon contact.  Causes severe tissue damage with prolonged contact.  May be harmful if inhaled.

 Keep containers tightly closed.  Avoid skin and eye contact by wearing all necessary protective equipment, including eye, face and hand protection and protective clothing.  Avoid inhaling by using in well- ventilated areas only and/or wearing the proper respiratory equipment.

 The material is very unstable. May react with water to release a toxic or flammable gas.  May explode if shock, friction or increase in temperature.  May explode if heated when in a closed container.  Undergoes vigorous polymerization.

 Keep material away from heat.  Open containers carefully; do not drop.  Store the material in a designated cool, flameproof area.

LIST OF HAZARDS FOR LAB 1 Escherichia coli Escherichia coli (E. coli) are bacteria found in a wide range of environments, including the digestive tracts of humans and animals. Most strains of E. coli are harmless, but precautions should still be taken to prevent unnecessary exposure risks. When working with E. coli in the lab, follow these personal safety tips:   

Treat all bacteria as potential pathogens Wear gloves when handling bacteria Thoroughly wash your hands after your experiments

The E. coli strains you will be working with today are considered a Biosafety Level 1 risk, meaning they pose little to no threat of infection to healthy adults. Work involving Biosafety Level 1 microbes can be completed at a lab bench and standard microbiological practices can be followed: (https://tinyurl.com/ybwst8js). BIOL301

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Fire Part of today’s experiment involves using a Bunsen burner. Bunsen burners produce an open flame and burn at high temperatures, meaning they are a fire hazard. When working with Bunsen burners in the lab, follow these safety guidelines:      

Remove all combustible materials (e.g., notebooks) from the area Tie back long hair and remove any dangling jewelry Do not wear gloves when lighting the Bunsen burner Keep your hands away from the flame Do not leave open flames unattended Shut off the gas valve when you are finished using the Bunsen burner, and double check that the gas valve is off before leaving the laboratory LABORATORY EQUIPMENT

Equipment is an integral part of any lab experiment. The equipment used for a given experiment depends on its purpose. Some types maintain samples at specific temperatures, either for growth (e.g., for bacteria), or for a reaction (e.g., restriction enzyme digests), while others are used to mix solutions or to isolate components from a solution. You will be encountering the following pieces of equipment in Lab 1 and in subsequent labs: Centrifuge Centrifuges are used to separate complex mixtures into distinct components based on material density. They rotate at high speeds, producing a centripetal force that separates individual components of a complex mixture by sedimentation rate, with denser materials pushed to the bottom of the tube and less dense materials remaining up top. How fast you set your centrifuge to rotate depends on what materials you are trying to separate. Today you will use the centrifuge to practice separating samples of different densities.

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A vortex is used to mix small volumes and functions by rotating at a set speed. When a sample is placed on the vortex, the vortex’s oscillation transfers to the sample. Vortexing ensures that a sample or solution is homogenously mixed. Today you will use the vortex to resuspend pelleted samples in solution.

t

Vortex

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Digital Scale Digital scales are used to precisely measure weight or mass. To use them, place a holding container (usually a plastic weighing boat) on top of the scale. Tare (zero) the scale to remove the weight of the weighing boat. Measure in the solid or liquid you want to weigh and record the weight that appears on the scale’s digital window. Today you will use the scale to measure out different volumes of a solution.

Incubator https ://www.fishersci.co.uk/shop/products/incubato r-

An air incubator is used to grow or maintain organisms at a set temperature. The optimum temperature for growth of bacterial strains is 37˚C, which is higher than the ambient room temperature of the lab. In the case of liquid cultures of bacteria, the incubator usually has a shaking platform to aerate the medium and clamps to hold the flasks firmly in place. You will be using incubators today to grow colonies of transformed E. coli.

Water Bath Water is a good thermal conductor. As such, a water bath is used to rapidly bring small sample volumes to a specific temperature and maintain them at that temperature. Small volume reactions such as restriction digests (37˚C) and ligations (16-18˚C) are generally done in water baths. You will be using a water bath today for heat shocking E. coli. https:/ /www.overstocklabequipment.co m/Fisher-Scientific-Isotemp-228-Digital-Wa ter-Bath

Bunsen Burner Bunsen burners produce an open gas flame used in a wide range of experiments. Bunsen burners are attached to a gas valve through a plastic hose. The gas is turned on, the burner is lit, and a blue flame is produced. Today you will be using a Bunsen burner to create a sterile environment for working with E. coli. https://www philipharris co uk/blog/secondary/bunsen burner

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Rotary Table A rotary table is used to help evenly spread a bacterial sample across an agar plate. The liquid sample is pipetted onto the plate, and the table is slowly turned while using a spreader to gently spread the sample across the agar. Today you will be using a rotary table to spread bacteria samples onto agar plates. Liquid Handling Experiments often require transferring small volumes of liquid tube/flask/bottle to another. There are two main ways of transferring liquids:

from one

1) Serological pipet: usually used for volumes of 1-50mL 2) Micropipettor: used for volumes of 0.1-1000µl Serological pipets may be plastic or glass and are calibrated with markings on the side of the barrel. They require a pump or bulb to draw and expel liquid from the pipet.

In contrast, micropipettes make use of a “pipet tip” that is appropriate for a specific volume range. Like serological pipettes, micropipettes are also restricted to a volume range. For example, a P1000 can transfer volumes of ~200µl to 1000µl but cannot accurately transfer volumes less than 200µl. Even more important, attempting to transfer a volume greater than the maximal volume will break a micropipette. Range of Gilson micropipettors used in BIOL301 (recall 1000µl= 1mL): Name

Volume Range

P20

2 - 20µl

P200

20 - 200µl

P1000

200 - 1000µl

Computer Each station will be given a MacBook Pro laptop computer to use during the lab (MacOS is High Sierra 10.13.6v). For those who are not familiar with how Macs work, ask a TA for help at the beginning of the lab.

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INTRODUCTION TO MOLECULAR BIOLOGY AND BASIC LAB SKILLS Molecular biology research often involves using many different experimental techniques and combining the results to arrive at a plausible explanation for why a particular phenomenon is occurring. This means that not only must a researcher be able to perform a variety of techniques, they also need to understand the purpose of each experiment so that they can correctly interpret their results and fit those results into a “bigger picture” explanation. For example, the end result of today’s experiment isn’t just to transform plasmid DNA into E. coli; it is to assess which of the two E. coli strains you are using is better at expressing proteins. This is an important question to answer for designing further experiments involving protein expression in E. coli. Today in Lab 1 you will be introduced to some basic laboratory skills that you will require to complete experiments throughout the semester. It is important that you learn these skills properly, so take your time as you go through the exercises and ask TAs for help if you are stuck or need clarification. Once you have had some practice, you and your lab partner will use your new lab skills in an experiment to transform plasmid DNA into E. coli as a starting point for examining protein expression in different E. coli strains. Learning Outcomes for Lab 1 Technical Outcomes Today you will learn how to:  Use micropipettors (P1000, P200, P20)  Use a tabletop centrifuge  Load samples on an agarose gel  Work with bacteria and use aseptic technique  Transform bacterial cells using the heat shock method Conceptual Outcomes  Understand the process of DNA transformation  Understand that E. coli strains differ in their ability to replicate DNA  Predict the expected outcomes of today’s transformation experiment Pre-Lab 1 Preparation Videos The following videos will help you to prepare for today’s exercises and experiments: 

Centrifuging (technique) https://www.youtube.com/watch?v=IhJNFGfsUus



Micropipetting (technique) https://www.youtube.com/watch?v=uEy_NGDfo_8



Agarose gel loading (technique) – watch up to 3min35sec



Plate Sp...