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Zamanian Lab Manual v 3.0

I. Lab Mission and Philosophy

A. Research Mission

The central ambition of our laboratory is to combine molecular biology, genetics, and computational approaches to improve our understanding of helminth biology and our ability to treat globally important human and animal parasitic infections. Current lab projects include the study of vector and soil-transmitted helminths at the bench and in disease-endemic settings. To complement some of our efforts, we exploit the experimental tractability of the model nematodes. We strive to place all of our work in the broader context of host-pathogen interactions and a growing appreciation for parasite genetic diversity and innovation.

B. Philosophy

We look to recruit individuals who share our curiosity and who wish to challenge themselves as they grow their scientific independence. We welcome students and trainees from a variety of backgrounds and with different career ambitions. I take my role as a mentor and advisor very seriously and it is therefore important to me that all members of the lab: (1) remain excited about the science we do, (2) challenge themselves to improve their base of knowledge and technical skills, and (3) engage in meaningful self-reflection about their progress and goals.

Borrowing from Federico Ardila-Mantilla, we build our lab upon these axioms:

  • Axiom 1. Scientific “talent” is distributed equally among different groups, irrespective of geographic, demographic, and economic boundaries.

  • Axiom 2. Everyone can have joyful, meaningful, and empowering scientific experiences.

  • Axiom 3. Science is a powerful, malleable tool that can be shaped and used differently by various communities to serve their needs.

  • Axiom 4. Everyone deserves to be treated with dignity and respect.

C. Mentorship

We hold weekly lab meetings as a springboard for open and critical scientific exchange. I will also schedule regular individual meetings with every member of the lab so that we can discuss research progress and whatever else that may arise. Career development is a multi-faceted endeavor. Where I am positioned to do so, I will identify or help create opportunities that relate to the career aspirations of individual lab members. I will work with trainees to help them hone their scientific writing and communication skills. This will occur through the drafting of manuscripts, as well as the attendance of relevant conferences. Once lab members have a firm grasp of their primary research projects, I will often encourage them to move out of their comfort zones. For many, this may mean achieving fluency in a programming language useful to their research and future. Lab members should also seek to make use of the expertise of the broader lab and university community. In general, your resources should include a wide network of peers and mentors. Foster relationships with others in your same position and those who are willing to offer advice. It is critical in science to foster your own support network of people you trust, are inspired by, and that have expertise you don’t yet have.

D. Lab Citizenship

My commitment to your success in research and professional growth is coupled to certain expectations. I expect all members of the lab to take ownership of their projects. I expect members of the lab to lend their help and expertise to one another and understand that our laboratory is a team with common scientific goals. I expect everyone to display good lab citizenship. We place value in an open, collaborative, and safe scientific environment.

1. Be helpful to others - this is a team effort!
2. Carry out your lab tasks in a timely manner.
3. Follow rules for ordering and inventorying items.
4. Keep your items confined to assigned spaces.
5. Do not borrow things without asking.
6. Clean up after yourselves and put things back in assigned locations.
7. Be here during normal work hours so that you can maximize interactions
8. Follow all rules when using facilities or resources outside of our lab.
9. Plan the use of parasites many weeks in advance.
10. Make sure the lab is properly shut down and communicate before leaving.

Lab dress code: For your safety, wear long pants or skirts and closed toed shoes, and tie back long hair. Do not wear shorts, short skirts, sandals, loose clothing, or dangling jewelry. Throughout your scientific career, you will be professionally interacting with people from diverse backgrounds and with diverse beliefs. In the interests of maintaining an inclusive and welcoming lab environment, I ask that you refrain from wearing articles of clothing that are likely to be perceived as hostile to another person’s heritage, ethnicity, gender, religious beliefs, or sexuality.

II. Communication and Calendar

A. Slack

All members of the lab will be asked to install Slack on their computers and phones and will receive an invitation to join the official lab Slack team zamanian-lab.slack.com. Slack will provide a basis for rapid individual and group communication on all research and lab-related matters.

B. Google Calendar and Group

An official lab Google calendar will be shared and updated with lab meeting times, relevant departmental and university events, and one-on-one meeting times. Lab members who will be out of town should notify me in advance and mark those dates on the lab calendar so that other lab members are aware should anything arise. Approved vacation days and other time off should also be entered into the lab calendar. You will also be added to the lab Google Group which will provide you access to Lab GDrive docs and sheets.

III. Research Documentation

A. Physical Lab Notebooks

All members of the lab should record the details of their daily research activities in the form of a lab notebook with dated entries. Lab notebooks must be turned in at the conclusion of your tenure in the lab. You have the option of using permanent lab notebooks or reusable notebooks by RocketBook to document your daily experimental activities. If you choose the RocketBook option, install the app on your phone and associate the account with your official university e-mail address. You should regularly take images and export your RocketBook notes to your dedicated folder on the lab Box (/RocketBook/) and to your Google Drive account as personal backup.

B. Electronic Lab Notebooks

The laboratory uses Benchling as a shared electronic lab notebook (ELN) and central repository for all molecular biology and cloning efforts. Lab members will receive an invitation to join the ZamanianLab Benchling organization page. Electronic summaries of work (including gels and printouts) should be regularly annotated and recorded into your defined Benchling lab notebook project space. This means that your electronic notes should include the minimal set of notes required to repeat any molecular biology experiment that you have performed. Do not let too much time elapse before you convert your physical notes into electronic form, as it is easier to clear up ambiguities when experiments are fresh in memory. This should develop into a habit. Benchling also provides tools for the planning of molecular cloning tasks and the design of reagents. Our lab plasmid inventory can also be found on Benchling.

C. Box Storage

All grad students and postdocs should install the Box Drive application and receive an invitation to edit the ZamanianLab shared Box folder. All students and staff of the University of Wisconsin-Madison receive Box cloud storage at no cost through their university e-mail accounts. The shared folder will be used to store important lab data and documents (lightweight storage). DO NOT delete shared folders while your local computer is set to sync as this will delete data across all devices. Do not use spaces in file names, unnecessarily long file names, or vague file naming systems that obscure contents. Individual lab folders are provided for members of the lab to store their research-related documents in /LabMembers/. These individual folders should be used to preserve processed data, protocols in the process of optimization, writing (manuscripts, fellowships, and grants), and any computer programs or scripts written to carry out preliminary analyses. The uses of other Box folders are described in other sections. See Computational Guide and Pipelines for descriptions of other storage resources.

IV. Meetings

A. Lab Meetings

Lab meetings will be held once a week to discuss general lab matters and for one person to present their work. Everyone presenting in the rotation schedule should have an updated presentation file (dated YYYYMMDD) in their personal /LabMeetings/ Box subfolder to present on their assigned days. This presentation should contain your overall project goals, divided into sub-aims, as well as data and timelines for specific experiments completed and planned for the coming weeks. This is a space for all members of the lab to contribute to troubleshooting and to engage in constructive and healthy scientific criticism. We will also keep 'flex' spots in our lab meeting rotation for volunteer driven journal or code club activities.

B. One-on-One Meetings

I will hold weekly one-on-meetings with every graduate student and postdoc, except when I'm traveling or have other commitments that I cannot sideline. You should come prepared with organized summaries of the status of your project(s), to share data you’ve produced, and to outline your next set of planned experiments. Each trainee and I will share a Google Doc to keep track of weekly and long-term goals. We will use this time to troubleshoot and constructively dissect these data and plans. This is also an opportunity for us to discuss general issues and your career development goals.

V. Lab Tasks

Every lab member will be involved in scheduled lab maintenance in some capacity. This will include things like defined cleaning activities and making common buffers and reagents. Roles are divided between undergraduates and other lab staff. Task assignments are outlined in the Lab GDrive.

VI. Lab Inventory and Organization

A. Lab Inventory

The locations of all reagents and consumables will be stored on Quartzy. Lab members will be expected to return ALL items to their assigned locations after use. It is the responsibility of lab members to make sure the Quartzy inventory is up-to-date. Additional rules are outlined in subsequent sections for inventory of plasmids, bacteria, worm strains, oligos, and parasite materials.

B. Lab Ordering

Lab order requests should be made through the lab Quartzy account. Add your initials to the Notes section of any order you submit. After approval, orders will be placed using either ShopUW+ system or the lab purchasing card (P-card). It is the responsibility of every member of the lab to communicate when we are nearing the end of necessary supplies or reagents so that orders can be placed in a timely manner. Except for routine items (e.g., tips, tubes, and gloves), you should message the PI on Slack prior to placing an order on Quartzy to receive approval. Each request should be associated with a funding source in the Quartzy order form. Orders will be fulfilled on designated days of each week. When items arrive, they should be immediately marked as received, physically dated, and associated with a location on Quartzy! P-card ordering: Items will occasionally have to be ordered with the lab P-card. P-cards are also often used for conference registration. I expect lab trainees to process these orders and to place a pdf receipt (appropriately named) of their order in /LabOrdering/PCard_receipts to facilitate processing. Amazon orders will be placed by the PI.

C. Physical Organization

  • Space Assignments: Benches and pipettes will be assigned to each grad student and postdoc. Undergraduates should only use the spaces and pipettes of their lab mentor.

  • Storage: Each graduate student and postdoc will have their own rack spaces in the -20C and -80C freezers for personal storage. Each person is responsible for maintaining proper labeling and notes of their stored items. There are designated storage spaces for lab and commercial plasmids, various bacterials stocks, C. elegans strain stocks, and parasite materials across lab freezers.

VII. Waste Management

  • Autoclave bag trash: Remove bag, twist top, bend over and wrap clear packing tape around the top to seal. Autoclave bags should be placed in the Rm 2934 MERI bins. Replace autoclave bag with new bag labeled 'Zamanian Lab'.

  • Gloves: Non-contaminated gloves go in the blue glove recycling buckets. Full buckets can be emptied into the designated loading dock box for recycling. We reuse the blue buckets.

  • Cardboard benchtop tip boxes: These boxes are designed for pipette tip trash only. When box is full, tape top closed with clear packing tape. Full tip boxes can be placed by either sink in the labs until there is room to add it to a autoclave bag. They cannot go into the MERI bin unless they are in an autoclave bag. New tip trash boxes can be found assembled on a shelf in Rm 2961 or unassembled in Rm 2934.

  • Uncontaminated serological pipette trash boxes: There is an empty tip box in each lab room for these. When the box is full, tape closed with clear packing tape and place a green 'OK to trash sticker' on it with name and date filled out. These can then go into or next to our trash bins in the lab. Replace with new box.

  • Liquid biosamples: Decontaminate bioliquids (parasite, bacteria, cells, blood/serum, stool) with 10% bleach before washing down the drain.

  • Empty blue pipette tip boxes: Boxes can be found in the cabinet above the plate reader in Rm 2961. The clear plastic in the boxes are recyclable and should be placed in the blue recycling container in the lab.

  • Glass trash: Glass that cannot be recycled (including broken glass) goes in the cardboard box in Rm 2963. Small glass slides and capillary tubes can go in red sharps containers. Full red sharps containers get capped and can go in or next to MERI bin in Rm 2934. New red sharps containers will be ordered as needed.

  • Hazardous liquid waste: There are very specific containers for Halogenated and non-Halogenated hazardous waste. Please see paperwork and containers under the hood in Rm 2935. Some Hazardous waste requires a special pick up from Safety. This can be managed through Kathy.

  • Worm boxes: Dirty worm boxes are placed in on shelf labeled 'dirty worm boxes' in Rm 2935. Clean boxes are found on top of shelving unit in Rm 2935. Boxes should be changed out often.

  • Dirty glassware: Dirty glassware in Rm 2961 should be placed in the tub on the cart under the dishware cabinet. Dirty glassware in Rms 4206 and 4210 goes in or next to the sink. Glassware is cleaned in the dishwasher found in the autoclave room. You will need to be trained to use the glassware in order to access the dishwasher calendar for scheduling.

  • Empty cardboard boxes: Empty carboard boxes should be flattened and placed outside of the lab.

  • Packing material: Some packing material can be recycled (look for recycling triangles). Some packing peanuts dissolve in water. All Styrofoam must go in blue bin in the SVM N hallway! Some shipping boxes with Styrofoam inserts can be returned to the company. Fill out the flap on box top and place on table behind mailboxes with note for US Mail pickup.

  • Empty parasite boxes from UGA FR3: These need to be sent back via Federal Express ground shipping to address on paper in box.

VIII. Lab Stocks

Locations and descriptions of all lab worm strains, plasmids, bacteria, and oligos should be stored and updated on Quartzy and in defined spreadsheets where specified. The following protocols and naming conventions must be followed, and lab members are responsible for checking the lab database to avoid naming conflicts prior to placing any new orders and assigning new names. Bacteria, worm strains, and plasmids procured from external sources and with existing names should retain those names.

A. Oligo Stocks

Use IDT_oligos.xls in /LabOrdering/Oligos/ to enter oligo details for ordering. Check Quartzy to ensure you are using the next oligo number available. Provide complete information (Species, Oligo Pairing, Molecular Target, Amplicon Length, and Tm) within the spreadsheet order form. All oligo types (DNA primers, gRNAs) should be assigned an ID with the same naming convention. Orders will be placed by a designated lab member.

Oligo Name: oMZ####
Storage box labels (-20C): "Oligo Stocks #"
* Quartzy Update (Type = 'Oligo or Primer'):
    Item Name (=Oligo Name)
    Sequence
    Technical Details (Species, Target, Tm, Pairing)
    Location
  • Primers note: Make 100 uM lab stocks of each primer oligo and store in Oligo Stocks box (-20C). Add 10*Y (uL) volume to lyophilized primer, where Y is nmol yield to make 100 uM stocks. Keep 10 uM (1:10 of stock) dilutions of primers in your own personal boxes for personal use. Re-order the original stock primer before it runs out if it will be needed in the future.

B. Plasmids (Lab and Commercial)

Lab Plasmids are plasmids created or designed by our lab for long-term use and storage. Commercial (external) Plasmids are plasmids we have received as gifts or ordered from vendors.

Plasmid Name: pMZ#### (lab) or Original Name
Storage box labels (-80C & -20C):
    "Lab Plasmids #" or "Commercial Plasmids #"
Tube Counts: 1 copy (-80C) + 1 or more copies (-20C)
* Tube Labels
    Plasmid Name
    Concentration
    Date Frozen
* Quartzy Update (Type = 'Plasmids'):
    Item Name (=Plasmid Name)
    Amount In Stock (units)
    Location
    Date Frozen
    Resistance Marker
    Use (e.g., "gene X RNAi" or "C. elegans transgene")

Use the stock(s) in -20C for all molecular biology purposes. When -20C stocks have been depleted, transfer the backup stock from -80C to -20C, and inoculate liquid media with the frozen bacterial stock to replace the plasmid stock in -80C. All intermediary vectors using for cloning that are unlikely to be used for other purposes should be stored in personal boxes until no longer needed.

  • Plasmids note: All completed (inventoried) plasmid sequences should be deposited into the designated lab plasmid Benchling project. Additionally, plasmid sequences and image maps should be exported to the Box folder /LabInventory/Plasmid_Export/.

C. Transformed Bacteria

Storage box labels (-80C & N2):
  "Lab Plasmid Bacterial Stock #" and "Commercial Plasmid Bacterial Stock #"
Tube Counts: 2 copies (-80C) + 1 copy (N2)
* Tube Labels
    Plasmid Name
    Bacteria (eg., DH5a)
    Date Frozen
* Quartzy Update (Type = 'Transformed Bacteria'):
    Item Name (=Plasmid Name)
    Amount In Stock (units)
    Location
    Bacteria (eg., DH5a)
    Date Frozen
    Plasmid (=Plasmid name)
    Resistance Marker

D. Feeding (C. elegans) Bacteria

Boxes specific to each bacteria (e.g., OP50, HB101, NA22) used for C. elegans feeding should be stored in the designated area of the -80C, noted with how many tubes of each bacteria per box in Quartzy. Replace all bacterial stocks before using up the -80C stock.

Storage box labels (-80C):
  "Bacteria Name"
Tube Counts: Variable
* Tube Labels
    Bacteria (e.g., OP50)
    Date Frozen
* Quartzy Update (Type = 'Feeding Bacteria'):
    Item Name (=Bacteria Name)
    Amount In Stock (units)
    Location
    Date Frozen

E. Competent Bacteria

Boxes specific to each competent bacteria (DH5a, NE5a, JM109) should be stored in the designated area of the -80C, noted with how many tubes of each bacteria per box in Quartzy. Replace all bacterial stocks before using up the -80C stock.

Storage box labels (-80C):
  "Bacteria Name"
Tube Counts: Variable
* Tube Labels
    Bacteria (e.g., DH5a)
    Date Frozen
* Quartzy Update (Type = 'Competent Bacteria'):
    Item Name (=Bacteria Name)
    Location
    Date Frozen

IX. Caenorhabditis elegans Strains

A. Strain Creation

Only the lab manager (Kathy) is allowed to use the microinjection apparatus to create transgenic strains. It will be the responsibility of any person who wishes for a strain to be made to:

1. Coordinate with Kathy at least two weeks in advance
2. Provide necessary background strain for injection (if not N2)
3. Provide injection mixes in the Plasmids box in the top freezer in room 2961.
  - Mark the tube with the date and all plasmids included in the mix
  - Including any fluorescent or phenotypic marker that should be followed
4. Provide all required information in the inventory spreadsheet (see below)

It is your responsibility to monitor injected lines and select for stable lines and confirming genotypes (through PCR and other approaches). Provide Kathy with the F1 plates to chunk from and cryogenically preserve once you have decide to keep any transgenic lines.

B. Strain Inventory

Lab strain designation: ZAM# Lab allele designation: maz#

C. elegans strains we receive from CGC, as gifts from other labs, or that we create in the lab will be frozen by the lab manager. Five tubes will be frozen at once, with one of those tubes frozen in the Liquid Nitrogen Tank. Strains must be associated with COMPLETE genotypic information (e.g., Transgenes, Alleles, Strain Background) on Quartzy AND in the C. elegans Strain Database linked in the Lab GDrive. The injection mix components and concentrations used to generate the strain as well as any additional notes pertaining to the strain should also be documented in the spreadsheet.

C. Strain Maintenance

Maintain strains in your assigned areas of the 15/20C incubators, or above your bench if incubating at ambient temperature. Use assigned colors to label your plate boxes. Wrap old plates and dispose of contaminated plates regularly. Your contaminated plates (bacteria, fungi, or mites) can jeopardize the experiments of others! Request chunks or frozen strains with adequate notice. Strains should be re-thawed or chunked at least once every six months to avoid mutation accumulation. Update the Rm 2961 whiteboard with the strains you are actively maintaining. Boxes should be baked before use to propagate new strains.

X. Parasite and Insectary Operations

A. Parasite Schedule

We generate parasites through local infections and receive additional regular shipments of filarial parasites (B. malayi, B. pahangi, and D. immitis) from the NIH FR3 repository and schistosome-infected mice from the NIH SR3 repository. The Lab Manager will place external orders for many months ahead and the expected scheduling of all parasite stage and materials will be updated on the Parasite Schedule Sheet linked in the Lab GDrive. Keep track of both local and external parasite orders. Shipments do not always arrive as expected, but you should have a detailed list of future parasite needs on the spreadsheet. I will send out a FedEx confirmation once I receive it (typically one day in advance) on Slack.

B. Storage of General Parasite Materials

General use (non-experimental) parasite materials should be catalogued and stored in designated racks/boxes. There are individual boxes for B. malayi, B. pahangi, and D. immitis tissues and RNA in the -80C freezer. There is a single box for storage of parasite gDNA in the -20C freezer. The contents of all samples in these boxes should be inventoried for each species in the appropriate tab of the Parasite Tissue and Nucleic Acid Inventory sheet linked in the Lab GDrive. The spreadsheet should be immediately updated after placing any new tubes in these boxes. This catalog of materials allows us to plan qPCR, RNA-seq and other experiments.

* Tube Labels (combine the following into a single string)
  1) Species (First letter of genus + first letter of species)
  2) Date (YYYYMMDD)
  3) Sample Type (T=tissue, R=RNA, CD=cDNA, GD=gDNA)
  4) Number ID (T###)
  Example: Bm20190530.T001

C. Parasite Infections (Insectary)

The insectary is a powerful resource that comes with major responsibilities. All members of the lab should follow established safety protocols and basic lab etiquette when using this space. No member of the lab should carry out insectary operations and procedures without approval from both PIs (Bartholomay and Zamanian). Detailed protocols for insectary operations, filarial parasite infection, and L3 isolation can be found on our Protocols page. Mosquito strains available for parasite infection:

  • Ae. aegypti LVP strain: used for B. malayi and B. pahangi infections
  • Ae. aegypti SD strain: used for D. immitis infections

Strains must be ordered in advance for infection, accounting for potential die offs and typical L3 yields. Place Orders as instructed in the Insectary Protocol and update the Parasite Schedule sheet linked in the Lab GDrive to associate parasite batches with mosquito infections

Mosquito Carton Labels
  Mosquito spp.:
  Parasite spp.:
  Parasite Source:
  Date Infected:
  Titer:
  Extraction Date:

XI. Writing and Reference Management

To streamline collaborative writing and/or editing of manuscripts and fellowship applications, lab members should create Google Docs to share documents with me and others involved in the writing goal. Paperpile will be used to manage references. Paperpile is compatible with Google Docs. A lab license will be made available to those who require it. For those who prefer LaTeX over traditional word processing, OverLeaf is also an acceptable alternative to Google Docs. All final data, figures, scripts, and text associated with manuscript submissions should be organized on Box and eventually transferred to a manuscript-specific GitHub project.

XII. Lab Safety

All lab members must take the appropriate safety training before starting any lab work and to use appropriate PPE at all times. Lab biosafety protocols and procedures will also be provided in binder and electronic form (/LabSafety/). In the event of emergencies involving accident or injury, call 911 to be routed to the campus police.

  • Training required for everyone

    • "Biosafety Required Training" (EHS)

  • Animal-related training for IACUC-approved work

    • "Animal User Orientation" (RARC)
    • "Safety for Personnel with Animal Contact" (EHS)
    • "Animal Contact Risk Questionnaire (ACRQ)" (UHS)
    • Additional 1-in-1 Training Offered through RARC (e.g., Mouse Training)

  • For work with human cell lines and potentially infectious materials

    • "Biosafety 102: Bloodborne Pathogens for Laboratory and Research" (EHS)

  • BSL-2 work

    E-mail both the Bartholomay and Zamanian lab groups (bartlab at g-groups.wisc.edu and zamanian_lab at g-groups.wisc.edu) in advance of BSL2 work in the insectary space. Put up BSL-2 signs on doors and include location, time, and reminder for PPE.

    Example e-mail: "I will be performing bulk parasite extractions from infected LVP mosquitoes TOMORROW (11/28/20). I will be performing the extractions in the screened-in work space inside the insectary (room 322). The parasites I will be extracting are Brugia pahangi L3s, and are classified as a BSL-2 organism. I will be working from 9:30AM - 12:30 PM, and will have signs posted on the outside of the screened-in area. If you need to enter this area during this time, please mind the PPE requirements specified on the posted BSL-2 signs. If this is a conflict/problem for anybody, please contact me as soon as possible. Thank you."

XIII. Emergency Protocols

-80°C Freezer

  1. Do not open freezers until you are ready to transfer contents. Notify emergency freezer contacts by phone.

  2. SVM North has one backup -80°C’s immediately outside of the freezer bay.

  3. Use the autoclave bins or empty styrofoam containers to collect dry ice and to keep items cold during transfer. The dry ice is located in the corridor outside of the freezer bay.

  4. Fill the bottom of your autoclave bins with the dry ice. Transfer items to the bins before carrying to the backup -80°Cs. Take boxes out of the metal racks if there is limited space.

  5. Record the date you transferred items on the backup freezer sheet.

-20°C Freezers and Refrigerators

  • If the -20°C breaks down or needs defrosting, coordinate with another SVM N lab for temporary storage space. We can also use a backup -20°C in Hanson if needed. Fill our autoclave bins with dry ice (located in the hallway outside of Rm 2936) to move items between freezers. It may be necessary to remove boxes from racks to fit everything.

  • If one of our fridges breaks down, transfer the contents to another lab fridge.

XIV. Lab Onboarding Checklist

  1. Room key and building card access form
  2. Office space, bench, and pipette assignment
  3. Computer assignment and environment setup
  4. Electronic accounts (will use UW email)
    • Google Group/Calendar (for shared sheets)
    • Box
    • Benchling
    • Paperpile
    • GitHub
    • CHTC
    • Quartzy (shared account)
    • DockerHub (if needed)
  5. Lab notebook (Rocketbook)
  6. Biosafety Training
  7. Photo for lab website