Molecular Phylogenetics Laboratory
MPL User Guidelines
The Molecular Phylogenetics Laboratory (MPL) is a research facility administered 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 VLSB. A lab manager in consultation with the faculty 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 Manager: Bridget Wessa, Office: 1129 VLSB, no phone
Molecular Phylogenetics Laboratory
I. BEGINNING AND ENDING YOUR WORK IN THE MPL
User Introduction and "Mentoring" Program
Per prior arrangement with a designated mentor, each new user must complete:
Undergraduates and Volunteers
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.
Ending Your Work in the MPL
Exit Inventories: Before any researcher leaves the lab, they should go through an Exit Inventory with their lab mentor/PI group and the MPL lab manager. All 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. With the help of the lab manager and group mentors, each lab group is responsible for processing their leftover kits, chemicals and group-specific hazardous material out of the MPL with their own chartstrings linked to specific funds.
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 Yellow 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 outlines additional health and safety information present in the lab 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 notebooks 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. Please store your caffeinated drinks, water bottles or other drinks, outside the MPL.
The lab manager is responsible for taking care of the requirements for two lab inspections each calendar year and a chemical inventory, which is maintained through an on-line 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
III. GENERAL LAB ORGANIZATION
The MPL is arranged as a series of common-use workstations designed for particular applications. No individual user has his/her own bench space for continual use. 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 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 Annex (LSA) Storeroom in LSA 171, or directly from vendors such as Fisher or VWR Scientific. Non-LSA storeroom orders are processed through the Berkeley Natural History Museums (BNHMs) Admin., which is currently Research Enterprises Services (RES). The lab manager enters purchase requests on-line in the BearBuy system and the Director authorizes the requests.
Note that the LSA Storeroom charges an overhead of currently 14%. They can purchase in bulk, so they often do get better pricing than individuals can procure even without the 14%.
Consumable Laboratory Materials and Services Supplied by the MPL
Chemicals Supplied by the MPL
Liquids: Bath-clear algicide, Ethanol (bulk metal cans and molecular grade pints), Ethidium bromide, Ethylene glycol, Glycerol, isopropanol, Liqui-Nox Labware soap, soft-soap/handsoap, bleach.
EHS Disposal Supplied by the MPL
Items or Services NOT Supplied by the MPL
A Note about Chemicals
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 rack to drain and dry, with the following exceptions:
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.
If you use the Eagle Eye Gel Doc. System please do your fair share to properly empty the solid waste bucket from the fume hood and replace the new bags.
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.
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 three 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. Updated program lists will need to be printed out for the 9600, as this unit does not list programs by Lab group and program name, as the MJ units do. The penalty for repeated offenses of these sign-up/cancellation and use guidelines will be denial of certain or all lab privileges for a period of time determined by the PIs of the MPL.
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.
Ethidium Bromide staining of Agarose Gels:The MPL uses Ethidium Bromide stain to visualize Nucleic Acids run out in agarose gels. There are other stains available such as SYBR or GelRed, but until there is a joint PI decision to migrate to a different stain, EtBr is the stain used in the MPL. There are pros and cons to using any given stain. See the 2010 EH & S link below for more info:
EtBr should be treated as a possible mutagen, carcinogen and teratogen and great care should be used when handling the stains as these solutions are easily absorbed into the skin. Users should always wear protective gloves (and a lab coat if desired) and be careful not to spread any EtBr into uncontaminated lab areas or labware. EtBr solutions should never go down the sink drains.
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. If this room is unlocked everything inside is at risk. 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 administrator 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, again with special authorization on your card key.