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BeANs Lab 101 Survival Guides
Sierin Lim NTU Bioengineering
N1.3-B3-11 +65 6316 8966
[email protected] 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
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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
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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
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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
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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
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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
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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
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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
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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
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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?
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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
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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
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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)
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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
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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
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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
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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!
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"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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