Molecular Phylogenetics Laboratory

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MPL User Guidelines
version: 04/19/16 blw

The Molecular Phylogenetics Laboratory (MPL) is a research facility sponsored by the University & Jepson Herbaria, at the University of California, Berkeley. Only personnel affiliated with the above organization and sponsored by one of the faculty members (listed below) are authorized to work in this laboratory. The MPL is structured to support PCR and post-PCR applications for molecular-based evolutionary, phylogenetic, and systematics research. This includes generating PCR products in thermal cyclers, screening PCR products, cloning of PCR products, and preparing PCR products for sequencing or fragment analysis. All genomic extractions should be done in each individual PI's laboratory, or research unit facility, separate from the MPL to avoid PCR contamination.

The MPL is located in rooms 1123 and 1125 of the Valley Life Sciences Building (VLSB). A lab manager in consultation with the faculty Principal Investigators (PIs) currently administers the operation of the MPL. These guidelines are intended as a set of established rules for the operation of the laboratory. They are not meant to describe all the details of every procedure nor do they replace mandatory instruction in lab safety and general procedures by the lab manager and designated mentors prior to beginning work and during lab use.

MPL Faculty PIs:

David Ackerly — Curator of Ecology

Bruce Baldwin — Curator of the W.L. Jepson Herbarium

Paul Fine — Curator of Tropical Plants

Brent Mishler — Director, Herbaria; Curator of Bryophytes

Carl Rothfels — Curator of Ferns and Lycophytes

MPL Manager: Bridget Wessa, Office: 1129 VLSB, no phone
The best way to contact Bridget is via email at bridge_@berkeley.edu.

Molecular Phylogenetics Laboratory
1123 Valley Life Sciences Building
University of California Berkeley
510-643-2519 (no voicemail)
http://www.ucjeps.berkeley.edu/MPL/index.html

I. BEGINNING YOUR WORK IN THE MPL

User Introduction and "Mentoring" Program
All personnel using the MPL must be authorized to work in this facility by one of the faculty PIs listed above. This PI will ultimately be responsible for the activities of their personnel while working in the lab. Each new user (regardless of their lab work history) will have an experienced mentor from their lab group designated to teach the new user in their MPL specific research protocols and about the details of using the MPL. This mentor is responsible for providing training and overseeing the work of this user with the help of the lab manager. The mentor should be someone in the same PI's research group, preferably a postdoctoral associate, senior staff member, or senior graduate student, and someone who already has extensive training and expertise in the methodologies used in the MPL.

Each new user must complete:

1. Three online campus classes
   • Lab Safety Fundamentals
   • Hazardous Waste Program
   • Spill Response

2. An MPL health & safety orientation with the lab manager prior to beginning training with their mentor. This orientation will cover compliance requirements for lab safety in the MPL, lab operational guidelines, the organization of lab supplies, and general hazard issues present. The new user will review and sign a "Lab Orientation Checklist" and an "MPL Users Authorization Form" prior to beginning any work. These records are stored in the lab for review or reference. The framework for this User Form was created by Abby Moore and Diane Erwin in 2010.

3. Detailed training with a designated Mentor. Only when each new user has been trained to the satisfaction of their mentor and the lab manager may the new user begin working in the MPL without the mentor being ever-present in the lab. This generally means that the new user has completed any given protocol at least three times (successfully) before they are "checked-off" on that protocol. Mentors should review MPL rules and lab protocols with undergrads/volunteers or anyone who is away for a summer (or length of time) on their return, particularly if they are new to molecular biology.

Undergraduates and Volunteers
Additional rules apply to undergraduates and volunteers (UG-V) working in the MPL. UG-V are only allowed to work in the MPL from 8 am to 5 pm, Monday through Friday, so that VLSB staff personnel can be contacted immediately if needed.

Mentors and UG-V should coordinate their time in the MPL for each session. Even after training, the designated mentor must be physically present in the MPL or in close physical proximity to the lab and be available to the UG-V at any time they have questions. Cell phone proximity is not acceptable if the mentor cannot immediately go to the MPL if needed.

In addition to the User Authorization Form and the MPL Health & Safety orientation forms, volunteers should fill out the Waiver of Liability, Assumption of Risk and Indemnity Agreement Form. Below is more information from Campus Risk Management:

Laboratory access is governed by the campus policy found at http://campuspol.chance.berkeley.edu/policies/LabAccess.pdf.

Visitors and volunteers to labs should be thoroughly trained in safety. While in the labs, volunteers must be under the direct, on-site supervision of an employee acting in the course and scope of employment.

II. HEALTH/SAFETY AND COMPLIANCE

Emergency Contact Information: In case of emergencies or safety issues in the lab, a list of important Cal ER contact information is posted near the inside of the lab door by the telephone. The lab manager will update this information whenever there are changes.

Users should be aware that campus labs are required to be compliant to on-site, local, state and national agencies. This includes following rules and regulations for the MPL, for VLSB, Campus EH & S, the Berkeley Fire Marshall, The City of Berkeley, East Bay Municipal Utilities District (EBMUD), Cal-OSHA and the EPA.

Per Cal-OSHA (title 8 CCR. sec. 5191) a standard Campus Chemical Hygiene Plan ("CHP" or Rainbow flip chart) is posted and maintained for the MPL. All users should read this material and sign the MPL "CHP". The material within this packet should be reviewed annually. This CHP lists common health and safety protocol requirements in the MPL and lists health and safety resources on campus.

The Red Compliance binders by the phone contain User Authorizations and User contact information, training records, EH & S Fact Sheets, MSDS information, and lab inspection and chemical inventory records.

Food and drink are NOT to be consumed in the MPL. There is a chemical-, glove- and labware-free area for reading, working on lab devices or storing your personal gear in 1125, but food or drink items should not be consumed in this area or any other area of the MPL at any time.

The lab manager is responsible for taking care of the requirements for two lab inspections each calendar year, a chemical inventory, and training records, which are maintained through an online campus wide system. General hazardous waste is processed out of the lab through EH & S with appropriate chartstrings linked to specific funds.

Relevant Standard Operating Procedure (SOP) information is posted at workstations and protocol binders are available in the MPL for review or reference. The lab manager maintains and updates these binders as needed. Users are encouraged to ask their mentor and/or the lab manager whenever they are unsure of something.

Hazards Present in the MPL
* All personal should follow proper protocols and wear appropriate personal protective equipment when working with any of the following:

• Bunsen burners and flammable liquids (for cloning)
• Clorox Bleach — pH 12.6, strong oxidizer
• Electrophoresis power supplies
• Nucleic Acid Stains (Gel Red and SYBR Green I) — suspected mutagen
• Sharps Razor blades
• UV Exposure — protect your eyes

III. GENERAL LAB ORGANIZATION

The MPL is arranged as a series of common-use workstations designed for particular applications. Shelf, drawer, fridge and -20° C freezer space are available for each lab group's materials, but space is limited. Users must process obsolete samples, stocks and chemicals out of the MPL as soon as possible, within health and safety guidelines. Drawers of general stocks are labeled to indicate the items stored in them. Users are encouraged to return labware not being used to their respective locations as soon as possible after use so others can find them.

Laboratory Supplies in the MPL
The MPL purchases "Common Need" supplies ONLY. Consumable lab supplies are not to be removed from the MPL for use in other labs. A Supplies-Needed clipboard is kept in the lab near the door to indicate any chemicals or lab supplies that are in low supply and to note any needed repairs or other requests.

The following is a list of items that are provided for general use in the MPL, purchased using funds of the Herbaria. Many of these items are purchased from the Life Sciences Addition (LSA) Storeroom in LSA 171, or directly from vendors such as Fisher or VWR Scientific. The lab manager enters purchase requests online in the BearBuy system and the Director authorizes the requests.

Consumable Laboratory Materials and Services Supplied by the MPL
v 4.19.2013

• Gloves (Latex and Nitrile)
• Microcentrifuge tubes (0.5 and 1.5 ml)
• Disposable 15 and 50 ml conical tubes
• Plastic petri dishes and cell culture tubes (14 ml round bottom)
• Pipettor tips: p10, p20-200, p1000, p10ml (in bags only)
• Pipettor tips: multi-channel pipettor stack racks (Rainin)
• Kimwipes, paper towels, bench diapers/bench paper
• Parafilm (2 inch and 4 inch rolls)
• Labware cleaning brushes and sponges
• Felt tip lab markers, Bic pens, pencils
• Cellophane, autoclave and lab tape
• Foil, plastic/Saran wrap
• Hand soap, labware detergent, bleach
• Whatman weighing paper and weigh boats
• Razor blades and sharps containers
• Glass transfer pipets and bulbs
• Disposable 5 and 10 ml polystyrene pipets
• Working stocks of Nucleic Acid Stains (Gel Red and SYBR Green I)
• "Burn-up" boxes and bags for broken glass and Nucleic Acid Stain solid waste
• White Fiberboard Fridge/Freezer Storage boxes and inserts
• Molecular ladders: 100bp, 1kb, 1kb+, low and high mass
• Batteries for lab timers
• Service, maintenance and repair of 1123 and 1125 equipment, labware, pipettors.

Chemicals Supplied by the MPL
Solid chemicals: Agarose (General Use only), Bacto-Agar, Bacto-Tryptone, Bacto-Yeast Extract, Blue Dextran, Boric acid, Bromophenol Blue, EDTA, ethidium bromide, glucose, Luria broth base, Magnesium chloride, Potassium chloride, Sodium Bicarbonate, Sodium Chloride, Sucrose, Tris Base (or Trizma), Xylene cyanole FF.

Liquids: Bath-clear algicide, Ethanol (bulk metal cans and molecular grade pints), Ethylene glycol, Glycerol, isopropanol, Liqui-Nox Labware soap, soft-soap/handsoap, bleach.

EHS Disposal Supplied by the MPL
EH & S chemical waste disposal fees for Nucleic Acid Stains Solids and Sharps.

Items or Services NOT Supplied by the MPL

• Thin walled Thermal Cycler Tubes
• Racked Pipettor tips p10, p20-200 or p1000
• Any filter tips
• Distilled water
• 96 well plates or seals
• Any Enzymes (PCR, PCR clean-up, cloning, sequencing, digests)
• Any Molecular Kits (Qiagen, Zymoclean, Eppendorf, Millipore, MoBio, etc.)
• EH & S disposal fees for ANY Molecular Kits (liquids or solids)
• Cloning Kits
• Antibiotics and Specialty chemicals (IPTG, X-gal) for Cloning Protocols
• Chloroform, Phenol, DEPC, Formamide, isoamyl alcohol, SOC
• *Ultra fine, 3:1, NuSieve or other specialty Agarose
• Loading dye (other than the type supplied with the Molecular Ladders)*
• Disposable filters (115 ml and syringe/disc cellulose acetate filters)*
  *(After current stock is gone, but there is still quite a bit left)

A Note about Chemicals
The MPL will provide "Common Need" chemicals ONLY. Please check your protocols ahead of time and check with the MPL manager to see if a chemical stock is in the MPL or that you have to purchase it on your own. There is also a copy of the current Chemical Inventory in the red compliance binders by the phone.

Remember: All chemicals are a liability, so if you need to purchase something, get the smallest amount possible for your needs and know that any individually purchased chemicals are now the responsibility of you or your lab group to process out of the MPL when these chemicals are no longer needed or were left behind. Do not accept "freebie" chemicals or kits from other lab mates, other labs, at vendor shows or from Reps. unless you are positive you will use them.

As stated in the User Form, you must check in with the MPL Manager before you bring ANY chemical into the lab. We need to be sure we can provide proper storage and use of this item and the Campus Chemical Inventory must be updated. All Individually purchased chemicals and KITS must be labeled with your NAME, complete list of contents (if not already labeled) and the DATE.

IV. SOME SPECIFIC LABORATORY RULES AND USER RESPONSIBILITIES

The PIs request that all Users sign a "check-in/out" log when working in the MPL. Please sign the log if you are putting PCR tubes into a thermal cycler. If you are just signing up for a thermal cycler time slot, there is no need to sign the check in-/out log, because you are not actually using the MPL equipment yet.

Each user is required to make up name and contact information cards for use at the lab workstations to identify the lab equipment, materials and space they are using when they are not actually present.

Read the signage around the lab. It's there to help create fewer problems for everyone.

Each user is required to "rack" pipettor tips from bulk bags for their own use in the MPL (except for the multi-channel pipettor tips). Place a clean, large Kimwipe on the bench to aliquot out a portion of the bulk tips using clean gloves to avoid contaminating the remaining stock for downstream users.

The same care should be taken when aliquoting out any other group stocks, such as microfuge tubes or chemicals.

Each user is responsible for cleaning up after themselves each workday. Glassware should be washed and DI-rinsed before placing it in the dishware racks to drain and dry, with the following exceptions:

• Thermometers should be put directly back in their drawer after cleaning. These tend to break in the dish drains.
• The Electrophoresis Rigs tend to fall out of full dish drains and break. These are very expensive. For this reason, please DI-rinse the rigs after use and return them immediately to the center bench shelf.

When in doubt, ASK. Do not jeopardize your work or that of others.

In general, use the oldest stock first so it does not become obsolete. Latex, Nitrile and plastics become brittle over time. Chemicals degrade.

If you use 1X TBE for agarose gels, please do your fair share to make more 1X stock for the general use carboy when it's low.

When you notice that a supply is low, please request it on the supplies-needed list by the door or contact the lab manager; instead of letting it completely run out for downstream users.

Each user is expected to properly store, handle and dispose of all old and/or unneeded stocks, kits, chemicals or samples under their responsibility.

Users are requested to provide prior notification to the lab manager and/or faculty PIs of official visitors to the lab. Please do not bring your friends into the lab to "hang out" with you when you are working, unless they are authorized to work in the MPL.

The penalty for repeated offenses of MPL guidelines will result in denial of some or all lab privileges for a period of time determined by the PIs of the laboratory.

Ending Your Work in the MPL
Please check in with your mentor and the MPL manager when you plan on ending your work in the MPL, so user information can be updated and we can coordinate an Exit Inventory.

Exit Inventories: All remaining stocks, samples, kits and chemicals must be accounted for in this inventory and where possible should be processed out of the MPL under proper guidelines. If a lab mate is taking over a "hand-me-down" stock, sample, kit or chemical, the new person's name should be written on the material at that time and the new owner is now responsible for this material.

V. USE OF INSTRUMENTS AND LABWARE IN THE MPL

All lab users should be thoroughly trained by their mentor before using any of the equipment on their own. If you need additional information, please ask and/or refer to the File Box of manuals assembled for use in the MPL, on the clean bench in 1125. All information in these files should remain organized in this box and should be kept in the MPL for lab users access. The lab manager will make updates to this information as changes occur.

Group-specific protocol information is not listed in this user guide and should be covered in detail during training, by mentors.

Pipettors: Please take good care of the pipettors. They are our most important tools for successful research. Please do not use the pipettors outside their volume ranges. Please use proper technique to avoid contaminating pipettors or getting any liquids inside the shafts. Wipe down the outside of the pipettors with a water-damp Kimwipe if any chemicals are on the outside of the barrel. If you think there is contamination inside a pipettor, please check in with the lab manager to see if a thorough cleaning is needed. Only experienced users should attempt to take a pipettor apart to clean it. If you notice that a pipettor is not working correctly or accurately please let the MPL Manager know so they can check the unit and send it in for service as needed.

Thermal Cyclers: Monthly sign-up sheets are posted near each of the thermal cyclers. Users should be considerate of other lab users in scheduling use of the thermal cyclers, especially because these are the most frequently used equipment items in the MPL. Most cycling programs take from 3.5-4 hours to complete. Please do not sign up to use the thermal cyclers unless you are actually prepared to use them or sign up for extra time slots just to give yourself a time buffer. Please erase your name as soon as you know you will not use a time slot. Please do not sign up weeks in advance.

Do not leave your PCR tubes in a +4° C HOLD for longer than ~1-2 hours and especially do not leave tubes in a thermal cycler overnight (at any temperature). Leaving the tubes at a 4° C hold is very hard on the thermal cyclers and one of the main reasons they fail. Never allow paper sheets or other items to block the vents on the thermal cyclers. Users should EXIT programs before turning the power off on all thermal cyclers or downstream users get a "Power Failure" review-history message. Do not write anything on the tops of thin-walled tubes; the ink will transfer to the bottom of the heated lid surface. Occasionally the lab manager will need to organize a "clean-up" of the thermal cycler programs to remove old/obsolete files and free up space for new ones.

Bench-Top Eppendorf Centrifuges: The rotors should be allowed to dry out between uses. Rotors stored wet corrode much faster. The plastic inner lids should not be stored attached to the top of the rotors. Place the plastic lids on top of the units when not in use. If users generate debris inside the rotors from their work, they should wipe out the inside of the centrifuge and around the rotor with a damp paper towel or Kimwipes. If corrosive or exceptional debris or residues are present, the rotors can be removed using a special tool for complete cleaning. Check in with the lab manager in this case.

Dry Blocks: ALL the Heat block units in the MPL are designed to be used DRY. Do NOT add water to the blocks. Users risk electric shock and damage to the units by adding water. The Labnet Digital Unit is reserved for sensitive protocols requiring accurate and stable temperature controls as a priority (i.e., heat-shocking cells at 42° C for cloning) and will be calibrated to 42° C. Please allow ~45 minutes for the digital block to stabilize at your desired temperature. The farther away from 42° C the longer it will take to stabilize. Please put your name card in front of any blocks you wish to reserve and remember to turn the units OFF when you are done with your procedure. The older analog blocks require many hours to stabilize. Keep this in mind for the timing of your work.

Nucleic Acid Staining of Agarose Gels: The MPL uses Gel Red or SYBR Green I stains to visualize Nucleic Acids run out in agarose gels.

Gel Red and SYBR Green I should be treated as possible mutagens and care should be used when handling the stains as these solutions are easily absorbed into the skin. Users should always wear Personal Protective Equipment (PPE)

Dry and Wet Ice, Millipore Filtered H20 and Autoclaves: Dry ice is available for a fee from the LSA Storeroom. Wet ice is available in VLSB 1104, across the hall from the MPL. Our only key to 1104 is kept in the top drawer below the phone. To lock 1104, you must use the key; it will not lock automatically behind you. Please return this key to the MPL as soon as you are done using it.

VSLB 4188 and 5180 provide wet ice, glass washers, 0.2 μ filtered water and autoclaves. Please note that although common use rooms are open 24/7 in VLSB, users need card key access to enter the 4th and 5th floors after 5 pm on weekdays and for weekends. Please see the VLSB facilities manager for these authorizations. If a VLSB autoclave is not working or an alarm is going off, note the room number and call the VLSB facilities manager. If a repair is needed, this can take many days, so use another autoclave for immediate needs. Additional autoclaves are available in LSA 524. You will need access to LSA via the 5th floor bridge OR into LSA after 5 PM from the first floor via the West Gate circle entrance.