Zamanian Lab Manual v 2.0
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 (parasitic worm) biology and our ability to treat globally important human and animal parasitic infections. This includes discovering new drug targets, elucidating mechanisms of drug resistance, and developing new tools for parasite manipulation. Our current work involves the study of mosquito-borne and soil-transmitted nematode parasites, as well as snail (vector) biology in relation to schistosome control. To complement some of our efforts, we exploit the experimental tractability of the model nematode Caenorhabditis elegans. We strive to place all of our work in the context of a growing appreciation for and understanding of parasite genetic diversity.
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.
We will 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 (e.g., R or Python) 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
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
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.
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 traditional lab notebook with dated entries. This is a requirement for federally-funded research. Lab notebooks cannot leave the laboratory and must be turned in at the conclusion of your tenure in the lab.
B. Electronic Lab Notebooks
The laboratory uses Benchling as a shared electronic lab notebook (ELN) and central repository for all molecular biology efforts. Lab members will receive an invitation to join the ZamanianLab Benchling organization page. Electronic summaries of work done and data generated (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 molecular biology reagents.
C. Lab Electronic 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, scripts, and documents. 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. The primary storage folders are described below. The uses of other Box folders are described in subsequent sections.
/Data/This folder should be used to neatly preserve original files and raw data output from various lab and campus instruments (e.g., subfolders
/qPCR/), common assays and projects with small data footprints (e.g., subfolders
/RNAi_experiments/), as well as minimally processed 'big' data outputs (e.g., subfolder
/RNAseq/). Other subfolders will be created to represent different project-specific data types where necessary, with strict guidelines for file nomenclature. These folders have clear organization and naming conventions that need to followed.
/LabMembers/Individual lab folders are provided for members of the lab to store their research-related documents. 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.
A. Lab Meetings
Lab meetings will be held once a week. Every member of the lab should have an updated presentation file (dated
YYYYMMDD) in their personal
/LabMeetings/ subfolder. 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. You should be prepared to present your progress in this short format every week when called upon. This is a space for all members of the lab to contribute to troubleshooting and to engage in constructive and healthy scientific criticism.
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 member of the lab 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: Undergrad Tasks and Staff Tasks.
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 Quartzy. Lab orders can be placed after review and approval. Orders can be placed using either the UW MDS system or 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 for the lab manager to process.
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 of materials related to individual experiments. 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, and bend over and wrap clear packing tape around the top to seal. The bags go in the MERI bins in the loading dock. 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 box on the loading dock 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 230 or unassembled on the bench in the loading dock.
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 large trash bin by elevator. Replace with new box.
Regular floor trash bins: When full, tie off top and place in large trash bin by elevator. Replace with new bag found in drawer S2 in 223 or on bench in the loading dock.
Liquid bacterial, parasite or blood: Decontaminate with 10% bleach before washing down the drain.
Empty blue pipette tip boxes: Boxes are placed in autoclave tub in 230 by sink. The clear plastic in the boxes are recyclable and can go in the blue recycling container by elevator. Colored inserts are also recycled through a program with USA Scientific. Empty colored inserts go in empty pipette tip box in 235 for pick up by USA Scientific.
Glass trash: Glass that cannot be recycled goes in the cardboard box in 223 by fridge/freezer this includes broken glass as well. Small glass slides and capillary tubes can go in red sharps containers. Full cardboard boxes get taped up with clear packing tape and put in hall outside lab doors with OK to trash green sticker filled out on it. Replacement boxes are in 235. Full red sharps containers get capped and can go in or next to MERI bin on loading dock. New red sharps containers are found on loading dock.
Hazardous liquid waste: There are very specific containers for Halogenated and non-Halogenated hazardous waste. Please see paperwork and containers next to hood in 230. Some Hazardous waste requires a special pick up from Safety. This can be managed through Kathy.
Worm boxes: Dirty worm boxes are placed in drying oven in autoclave room for a few days. Clean boxes are found on top of shelving unit in picking room in 230. Boxes should be changed out often.
Dirty Glassware: Dirty glassware in 230 goes in tub on cart by the sink. Dirty glassware in 223 goes in or next to sink. Glassware is cleaned in the dishwasher in autoclave room on program #4.
Empty cardboard boxes: Empty carboard boxes go on top of blue mixed paper bin by the elevator.
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 Animal Science loading dock! Some shipping boxes with Styrofoam inserts can be returned to the company. Fill out 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
/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.
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
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 223 - 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 Lab Sheets. 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 from the lab manager with adequate notice. Strains should be re-thawed or chunked at least once every six months to avoid mutation accumulation. Update the Rm 230 whiteboard with the strains you are actively maintaining. Boxes should be baked before using them to propagate new strains.
X. Parasite and Insectary Operations
A. Parasite Schedule
We generate parasites through local infections and receive 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 Lab Sheets. 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) in the
#Parasite_Planning Slack channel.
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 Lab Sheets. 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 Bartholomay Lab 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. 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 Lab Sheets 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 within
/Manuscripts/ and in 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
For work with human cell lines and potentially infectious materials
- "Biosafety 102: Bloodborne Pathogens for Laboratory and Research" (EHS)
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:
Do not open it until you are ready to transfer the contents. Notify emergency contacts on freezers by phone.
Hanson has 2 up right backup -80°C’s in the basement room B37. One right when you walk in and the other off to the right both labeled "back up."
Use the autoclave bins and any empty styrofoam containers you can get (found in the little cubby at the entrance of room 230) for getting dry ice to keep items cold during transfer. The dry ice is located on the loading dock to the right in tan wheeled bins.
Fill the bottom of your autoclave bins with the dry ice. Transfer items to the bins before carrying them down to the basement -80°Cs. Take boxes out of the metal racks if there is limited space is in the back up freezers.
Record on the sheet when you put items in them.
If the -20°C breaks down or needs defrosting, temporarily use the Bartholomay Lab freezers in 311 or 322. Both freezers have items in them but our -20°C contents should fit.
Fill our autoclave bins with dry ice (located on the loading dock to the right in tan wheeled bins) 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 in one of our other fridges.
XIV. Lab Onboarding Checklist
- Room key and building card access form
- Office space, bench, and pipette assignment
- Computer assignment and environment setup
- Electronic accounts (will use UW email)
- Google Calendar
- Google Group (for shared sheets)
- Biosafety Training
- Provide photo for lab website