BeANs Lab 101 Survival Guides

Sierin Lim NTU Bioengineering

N1.3-B3-11 +65 6316 8966

SLim@ntu.edu.sg http://www.ntu.edu.sg/home/slim

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The Expectations

Hours • 10 (UG) – 50 (PG) hours/week in the lab (minimum) • 2-3 hours reading

Log book

• Maintain daily journal • Include objective, all protocols, results, and conclusions

Update • Weekly • Attendance at group meetings

Present • Once per semester • 30-40 min

Expand • All project objectives are expandable

Data • Publishable • Ph.D. = > 3 papers; M.Eng./M.Sc. = > 1; B.Eng./B.S. = > 0.5

GAME RULES

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General Rules • Be safe – it is your responsibility!

– Completion of the SCBE safety training is mandatory prior to any lab work – Wear necessary protections (goggles, gloves, etc.) – Dispose of sharps and toxic wastes in the designated containers

• Be considerate to others – We are part of a whole; take care of each other – Do not hog on an equipment – Clean up after yourself

• Always communicate – If you don’t know how/what to do, always ask others. It’s better safe than sorry – If you have concerns, do communicate them. Misunderstandings only lead to

unnecessary frictions.

• Take good care of the lab • If anything goes wrong in the lab, your work (and degree) will be affected

Computer Usage

• This computer is only for research purposes

• You can save your work in a folder with your name under “DOCUMENTS” – Path: C:\Users\Lim Lab\Documents\YourName

• Documents will be backed up at least twice a

year • Please keep a backup of your data at all times

Lab Database

• Most lab documents (protocols, recipes, past presentations) are available on – http://docs.google.com

• Lab wiki: – http://mysites.ntu.edu.sg/slim/BeANsLab

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Lab Meetings

• Aimed as update and trouble shooting sessions

• Held once a week unless otherwise mentioned

• Each member will give weekly update – 1-2 powerpoint slides

• One member will give a formal presentation each

week – Sign-up at the beginning of the semester – Can be in a form of journal club or own research

PRESENTATION GUIDELINES

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Things to keep in mind

• Spend a few days to think about the flow of your slides; and only a few hours to make the slides. – What is the story that you are planning to tell?

• KISS: Keep It [the story] Simple and Sweet – Do not tell me that you cannot work in the lab because you are

making slides for your presentation! • You should already have enough slides from your weekly summary

• Presenters are expected to be set-up 10-min prior to

meeting time.

• Key to the meeting/conference room can be obtained from the BIE (N1.3-B5) or CBE general office (N1.2-B3): – Check website for actual details

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WEEKLY MEETING

All data are to be presented in figures/tables complete with captions

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Progress for this week’s experiment: 1. Experiment 1 2. Experiment 2 3. Experiment 3

The most important result:

Important result #1: Important result #2:

Name: Date:

Figure with caption

Short conclusion

Figure with caption

Short conclusion

Figure with caption

Short conclusion

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Other result #1: Other result #2:

Failed experiments: Next week’s plan: 1. 2. 3.

Summary

Why they fail What you will do to fix it

Figure with caption

Short conclusion

Figure with caption

Short conclusion

TERM MEETING

This is a good practice before the real-world presentation, so put some efforts to it

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OUTLINE OF PRESENTATION/INTRODUCTION

• SKIP THIS SLIDE – If you are just going to list Intro, Method, Results,

Discussion, etc.

• Give meaningful titles to your slides – i.e. the immediate goal of the experiment

• General rule of thumbs

– Timing: 1 slide/minute – Font: ≥18 points (any with equal width, e.g. Arial, Calibri,

Helvetica, Sans-serif, Verdana) – Colors: No or

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red on blue yellow on white

A general description of your project will be the title of your intro slide

• Depending on the length of your talk, the first 1-3 slides are the most general slides

• Describe the motivation for your project

– What is the big picture? – What others have done – What question/problem you are trying to

answer/solve

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Mention your system in the title of your background slide

• Overview of your system – The protein/material that you are working on

• Your hypothesis

– The basis of your hypothesis • How did you come to this hypothesis? • Is it based on previous reports? If so, what were they?

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Summary of your previous results

• In bullet points – Assess your progress – What you have done in the previous term – What problems you encountered – How it was solved (if applicable)

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Your objectives for this term

How you plan to achieve your objectives

• Brief and concise description of your methods • Include important details:

– Mutagenesis: • Primers, sites

– PCR: • Annealing temperatures, length of each step

– Gene expression experiments: • Host, growth temperature, inducer concentrations,

length of induction

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The objective of experiment #x will be the title for your result #x

• Mention the 3 take-home messages for each slide • Discussions should be concise • You can have as many slides as needed for this part

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Figure with caption

Short conclusion

Aesthetics is not everything but it is important

• Avoid misspellings

• NTU Powerpoint template: – http://www.ntu.edu.sg/AboutNTU/CorporateInfo/Cor

porateIdentity/UniversityIdentityGuidelines/Pages/Powerpoint.aspx

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“A picture is worth a thousand words” Avoid busy/wordy slides

Conclusions

• Simply summarize your findings

• State 5-6 important points that you want your audience remember

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Future works

• What you are planning to accomplish in the next few months – The exact experiments that you are going to

conduct – Anything that needs to be purchased

• Bring along quotations (if applicable)

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Acknowledgements

• Your mentor • Fellow lab mates who actually help you • Other friends/lab mates

• Funding agency:

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GOOD LAB PRACTICES

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Keeping a GOOD LAB NOTEBOOK is crucial

• Everyone should have and keep a good Lab Notebook that: – Is bound with numbered pages NO LOOSE LEAVES – Is updated daily – Contains ALL details of each experiment

• This routine will help you in troubleshooting and repeating the

experiments in the future

• Lab notebook will be examined every week during the lab meeting • The lab notebook will remain in the lab after you finish your work

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Can someone use my notes 5 years from now, do the same experiment, and expect the same result?

Literature review & cataloging references

• Spend the first couple of weeks reading literatures to understand the background of each project – Get the BIG picture and the WHY I am doing this

• Keep a library of all relevant references that you read in PDF file

• The suggested format for file name:

– [first-author-surname]-[corresponding-author-surname]-[publication-year]_[abbr-journal-title]_[TitleOfArticle]_ [OtherInfo]

• E.g. bretscher-thomson-83_emboj00257-0115_DistributionFerritinReceptorsCoatedPits _HeLaCells

• If you have access to EndNote, please use it to keep a library of your

references and attach the PDF file to each reference item

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Plan your experiment

• Suppose you are going to publish a paper, what figures should go into your paper?

• Spend 1-2 days prior to the actual experiment to think through how to obtain your data

• Do a quick check to make sure that you have all of the required materials the day before

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IN THE LAB – SAFETY FIRST YOUR LAB NOTEBOOK IS YOUR ABSOLUTE COMPANION

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SAFETY First

• Prior to any lab work, every member MUST complete the SCBE/university safety training

• Wear the appropriate personal protective equipment (PPE) • NO unattended open flame, especially near flammables • If you have long hair, please tie it back whenever you are working in the

lab

• All chemicals come with Materials Safety Datasheet (MSDS); please CHECK for any precaution before commencing work, for example: – Ethidium bromide (mutagen, carcinogen) – Ferricyanide (oxidant; highly toxic under strong acidic condition) – Acetone, methanol, ethanol (flammable)

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Chemical Safety

• Storage: – All flammable materials are stored in the yellow cabinet marked

with “FLAMMABLE” stickers • Draw only small/reasonable quantity from the stock each time

– Acid and base should be segregated and placed in secondary container (bin/bucket, etc.)

• Transporting liquids (particularly toxic ones): – Always have them in secondary container (bin/bucket, etc.)

• Disposal: – All wastes are to be disposed of in the designated container – See “Dealing with wastes” section for details

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To wear or not to wear

• LAB COAT – whenever you are in the lab – Monthly/bi-monthly laundry service for lab coats are available

• SHOES – must be close-toed • GOGGLES

– When excising gels on UV box, working with volatile compounds

• GLOVES

– Wear: whenever you are working with chemicals (e.g. ethidium bromide, cyanite, methyl viologens)

– DO NOT wear gloves when you • Touch computer keyboards, door knobs • Are walking around the hall way; If you absolutely need to wear them

while transporting something please have only wear glove on one hand

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The Lab Notebook • Start with a date • State a brief objective/hypothesis:

– What is the experiment trying to accomplish?

• Include ALL details – see next slide

• If you do data processing, include the print out and indicate the name of the file (softcopy)

• All print out should be taped/glued securely in the Lab Notebook

NO LOOSE LEAVES

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What to keep in a lab notebook

• Detailed protocols (only need to be done once; ref page# for future) • Any changes /deviation that you made from the original protocol • Batch number of each sample • Experimental parameters (host, [concentration], volume, length of

incubation, temperature, pH, etc.) • RAW DATA and detailed calculations • Any observations

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Write down everything! Ideas, thoughts, color, precipitate, duration, etc.

Small changes may have BIG implications

Protocol development

• A recipe for a cookbook

• Write down – The “ingredients”, including:

• Stock concentration • Volume added • Volume of reaction • Temperature • Start/end time

– The“1-2-3, step-by-step” that you do/did

• A good protocol will be transferred to the lab template

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Experimental results must be REPEATABLE & RELIABLE

• There is a difference between measurement variation and experimental variation – Measurement variation – within the same batch – Experimental variation – between different batches

• Each measurement should be repeated at least THRICE

with variation of 5-10%

• Each experiment should be done at least in duplicate

• The best practice will be to take an average of means and report the standard deviation of the mean

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What to repeat and how many times

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Experiment 1

• Measurement 1 • Measurement 2 • Measurement 3

• Average 1

Experiment 2

• Measurement 1 • Measurement 2 • Measurement 3

• Average 2

Experiment X

• Measurement 1 • Measurement 2 • Measurement 3

• Average X

Average ± STD of average UNIT

CONTROLS are important! • Positive control

– How it should work • Negative control

– How it should NOT work

• For PCR: – Plasmid without gene of interest (-) – Other reaction with unrelated gene and

its primer (+) • For digestion:

– Uncut plasmid during electrophoresis (-) • For ligation:

– One reaction with water as the insert (-) • For expression:

– Untransformed cells (-) – Uninduced culture (-); will tell you

whether your expression is leaky/not – Other gene/host that is known to produce

protein (+)

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LABEL and DATE all your samples • VERY IMPORTANT

– Unlabeled/poorly labeled samples are subject to clearance (i.e. trashed)

• What to include: – Sample name – Date – Your initial

• Samples include:

– Solutions of antibiotic/media/buffers, etc.

• Protein samples – Batch number is the date you break the cells

• E.g. If you do french press/sonication of cell pellets on 1 May 2011, then the batch number of your protein is #010511

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Your samples are precious

• NEVER throw away your samples unless: – They are spoilt (contaminated/precipitated)

• Some “old” samples may:

– need to be revisited – provide valuable clues – be the seed of discoveries

• Suggestions: keep all samples until the annual lab

“Spring cleaning” exercise

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Lab inventory • Update the softcopies on googledocs and lab computer

– When there is addition/removal from the inventory

• Chemicals/kits – Date received, Date opened, Your initial – Include abbreviation/common names, e.g. 3-(3,4-dichlorophenyl)-1,1-

dimethylurea (DCMU)

• Strain Master Stock – contains all the plasmid/cells that you can always go back to – Stock number (both on top of the tube and on the side label) – Name of stock: [plasmid-name]/[host], e.g. pE2/DH5α – Date, Your initial

• Plasmids & Plasmid Maps – Name of stock, Date, Your initial

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Glass & plastic wares

Glass • To label the content, date,

owner: Use marker – Marker can be rinsed off

using ethanol (EtOH) or acetone (Ac)

• Suitable for use with many

solvents

Plastic • To label the content, date,

owner: Use tape & marker

• Do not use bleach or organic solvents with PC/PET/PS

• Do not use PE/PP to make acid/base stock – Due to its porous nature,

residues cannot be removed easily

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Types of plastic Name Clarity Notes

Polycarbonate (PC) Clear No bleach/organic solvent/Ac

Polystyrene (PS) Clear/ Opaque

No bleach/organic solvent/Ac

Polyethylene terephthalate (PET) Clear No bleach/organic solvent/Ac

Polyethylene (PE): high/low-density Opaque OK for Ac, NO for acid/base

Polypropylene (PP) Opaque OK for Ac, NO for acid/base

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Glass PC (spoilt)

Solution concentrations

• Working concentration ([working])= 1x • Concentrated solutions = Ax

– 10x solution means the solution is 10 time more concentrated than the working concentration

– To use it: C1V1 = C2V2 • Dilute it 10x to achieve 1x • 1 part concentrated solution + 9 parts water

• Concentration is often expressed as % (w/v)

– 10% = 10 g/100 ml 43

Water types

• Type 3 water is the lowest laboratory water grade, recommended for glassware rinsing, heating baths and filling autoclaves, or to feed Type 1 lab water systems.

• Type 2 water is the grade used in general laboratory applications such as:

– Buffers, pH solutions and microbiological culture media preparation; – As feed to Type 1 water systems, clinical analyzers, cell culture incubators and weatherometers; – Preparation of reagents for chemical analysis or synthesis.

• Type 1 water is the grade required for critical laboratory applications such as:

– HPLC mobile phase preparation, blanks and sample dilution in GC, HPLC, AA, ICP-MS and other advanced analytical techniques;

– Preparation of buffers and culture media for mammalian cell culture and IVF; – Production of reagents for molecular biology applications (DNA sequencing, PCR); – Preparation of solutions for electrophoresis and blotting.

• Using Type 1 water for Type 2 water applications is a common laboratory practice

in order to decrease the risk of artifact generation during experimental procedures.

Source: http://www.millipore.com/lab_water/clw4/tutorial&tabno=4 44

Making solutions (1) • Calculate the amount of solids/liquid required to make solution of a

certain concentration – Mass (g) = [solution] (M) x Volume (L) x Mol. Mass (g/mol)

• Weigh out the salt solids; Make sure that the balance has at least one

extra digit from the amount you are measuring – E.g. you want to measure 1 mg; the balance should be accurate to at least 0.1

mg (=0.0001 g)

• Place salt solids in beaker; Add Type 2 water to the salt solid slightly less than the final volume (volume may change when the solids are dissolved)

• Stir and adjust the pH using either HCl (4 M) or NaOH (4 M) • Top-up and adjust to final volume with graduated cylinder • Check pH once more

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Making solutions (2)

• For DNA/protein work, filter with 0.22 or 0.45 µm filter to remove debris and bacteria

• For purification, degas (apply vacuum while stirring) for 20 min.

• Acid over water; not the other way around – Adding water to acid is usually

exothermic and may cause explosion

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Aseptic technique • AVOID contamination at all cost!

– Perform experiments/transfer in biosafety cabinet (BSC) and/or in the presence of flame

• Bacterial cell culture

– Transfers easily – Between samples:

• If you are using a metal inoculation loop, make sure you flame it until it is red, dip it in agar to cool

• Insect & mammalian cell culture

– Easily contaminated – take good care during experiment – Always wear gloves and have a spray bottle containing 70% EtOH ready – Prior to each experiment: wipe down the surrounding workplace – Prior to handling sample: spray your gloves and media/culture flasks

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Making strain stock • A good Stock is the backbone of a life-long research

• How-to:

– From a single colony, grow 7-ml overnight (O/N) culture – Make double stock; for each stock:

• Mix 900 µl O/N culture and 600 µl 50% (v/v) sterile glycerol 20% (v/v) [final] • Label following slide #35, assign a temporary number • Store at -80°C

– Use the 5 ml for plasmid prep – Send for sequencing (1st Base) – Confirm sequence by sequence alignment (online: Needle)

• Once a plasmid construct is confirmed (i.e. no mutation), go to the

inventory list, get the actual number, and – Integrate ONE into the Strain Master Stock – Keep ONE in your box

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Plasmid maps

• Every plasmid made should have a map

• Online depository: https://benchling.com/

• Please make sure you know what you are doing and that the sequences are correct

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Dealing with wastes • Sharps and needles:

– Disposed of in the designated containers that is puncture proof (i.e. for SHARPS, cardboard box with taped seams and lined with plastic; for NEEDLES, plastic/metal container with lid

• Biological wastes: – Overnight plates: autoclave – Overnight culture: in receptacle containing 10% (v/v) bleach – Large/expression culture: add 10% (v/v) bleach and leave for at least

30 min., check pH, and dump into sink • NEVER dump untreated biological waste into the sink!

• Chemical wastes:

– Disposed of in the designated containers

• ALL liquid waste has to be neutralized (pH = 7.0 ± 0.5) before dumping into the sink, else alarm will go off – To check pH, please use pH paper (not the meter)

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Keep sink & lab benches clean & tidy

• The speaker of the week will be responsible in making sure that the lab is in its working order, clean, and tidy

• Washing labwares – Brush with soap – Rinse with tap water – Last rinse with DI water (type III) thrice

• Clean-up after each experiment

• Wash your hands before leaving the lab

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DATA PROCESSING

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EXCEL / WORD files

• This is not a replacement for the Lab Notebook

• Include the date of your experiment • Transfer your raw data and some details • Indicate the corresponding page number on the

Lab Notebook

• Print out the finalized graphs/figures, paste them on the Lab Notebook

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You can also include the equation here

Keep good records & notes

• Good data may be published even though not immediately

• You will forget the experimental details a few

days/weeks/months/years from now, so WRITE THEM DOWN!

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Publishing your research 101

• From George Whitesides on ACS Journal – http://pubs.acs.org/page/publish-research/episode-1.html

• Think of what figures you are planning to have on

your paper/report – This will help you in planning your experiments and

outline your paper – On average: 1 figure/printed page – 2000-2500 words (10-12 pages double spaced; 12

point Times New Roman; exclude references & figures) ~ 5 printed pages (including references and figures)

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Before you leave the BeANs Lab

• Please burn the following items (with proper annotations) onto a CD: – ALL softcopy of your raw and analyzed data, including:

• Sequencing and alignment results – ALL presentation files – ALL reports – ALL protocols that you developed

• Clean-up all samples during the annual “Spring cleaning”

– Pass confirmed constructs to permanent members of the lab – Trash failed samples

• Swing by B3-11 to drop off the CD, lab notebook, and have a little chat

about your future plan

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Get a celebratory dinner with the rest of the lab!

"Dans les champs de l'observation le hasard ne favorise que les esprits préparés." (In the fields of observation chance favors only the prepared mind.)

Louis Pasteur during a lecture at University of Lille (7 December 1854)

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Have a good time!

Learn hard Work hard Play hard

and at the end

It’ll all be worth it…

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