Lab Operations Checklist Lab(s): Room 317, 311 SSMB, College of Charleston Date Training Initiated: PI Name: Pam Riggs‐Gelasco Student Name: Student ID # Activity Description Student Date PI Mandatory initials complet‐ initials regardless of ed project Safety and Location of safety shower, eyewash, fire extinguisher, first aid kit, safety Industrial glasses, lab coat rack, MSDS sheets, spill kit, emergency contact X Hygiene information, updated lab cell phone list General departmental research training completion X Incident reporting guidelines and procedures X Monthly lab safety review and record‐keeping procedure X Policy for hand washing prior to eating X Policy for gloves wearing, when to take off gloves, what to touch with gloves on (with special note about the Gel Doc area, for which gloves are mandatory). No gloves on door knobs or phones. Not all gloves offer X equal protection; select proper gloves type for your particular experiment. Location of training records and guidelines X Environ‐ Waste disposal guidelines, especially for ethidium bromide, acrylamide, X mental and bacterial cultures Biohazard waste disposal procedure X Spill clean‐up kit location and procedure X Lab check list Both gas valves are off, and Bunsen burner valves are off. X before leaving lab each day X Fridges, freezers, ‐80, incubators, shakers, and centrifuges are all closed. Equipment used in 311 is secured, cleaned, and your own items returned to our lab: Autoclave, Sorvall centrifuge, Eppendorf centrifuge, X UV‐vis, PCR machine, MilliQ water system, etc. Temperature‐sensitive reagents and samples are properly incubated or X refrigerated or frozen according to their needs. Room‐temperature chemicals, pipet tip boxes and other sterile supplies, etc. are put away. Do not leave items on the benchtop that the X midnight cleaning crew could damage or contaminate. Dirty dishes are either clean/drying or have water in them so nothing X dries onto them overnight. Sliding glass doors covering the shelves are all closed. X Lab door is locked. X Night and Notify PI any day you plan to be in the lab when the PI is not in the X weekend building. operation Before coming to lab on nights/weekends in absence of the PI, confirm X that your experimental plans are suitable for working alone. Before leaving lab, double‐check the following: Gas is off, fridges/freezers/incubators/shakers/centrifuges are closed, autoclave X and ‐80 are okay, door is locked. sterile lab Common locations for storing sterile items: There are certain shelves items / House‐ and cabinets where all items inside are sealed and sterile. Sterile items keeping can be identified with autoclave tape with heat markings. If you contaminate a box of tips or other sterile item, remove the tape so X others don't think it is still sterile. Use autoclave tape on dry items.

Bacterial Contamination: Any disposable item such as a pipette tip that has touched bacterial culture needs to be autoclaved prior to disposal. X Glassware that has touched cultures need to be soaked in bleach solution and then rinsed thoroughly Glassware cleaning procedures for various items. No acetone. Final MilliQ water rinse. Do not let things dry onto glassware. X

Lab organization, equipment location, and cleanup protocols X Chemical Bacterial media. We keep on hand sterile aliquots of LB media in the storage refridgerator. Make more if you use the last one. X

Reagents for common use. Examples: gel prep and gel running buffers, etc. Don't contaminatethese and remake if empty. X

Storage of microbe plates upside‐down in fridge. Use marker codes for X presence of antibiotic Storage and labeling and record‐keeping of all cells, strains, mitochondria, etc. in the ‐80 freezer. X Storage of reagents in the refrigerator (4 degrees C), freezer (‐20 degrees C), and ultracold (‐80 degrees C) X Flammables/combustible cabinet location X Acid and base storage locations and containment X General reagent storage X Lab techniques Weighing out chemicals without any cross‐contamination. Make sure all spatualas are clean prior to using in reagent bottle. Do not return X anything to a reagent bottle. Caution when using toxic, carcinogenic, or poisonous chemicals: Protect skin/eyes, keep the reagent clean and contained to eliminate threat of X accidental ingestion, clean the balance area well after use. Caution when using concentrated acids/bases: Protect skin/eyes, keep X the balance area and containers clean. Caution when using DMSO: Protect skin/eyes. DMSO is carried through X skin. Ethidium bromide‐containing agarose gel heating, casting, transfer to X Gel Doc, disposal Acrylamide gel casting, handling, disposal X PCR reaction set‐up with emphasis on keeping reagents cold and not x cross‐contaminating them Sterile inoculation of liquid cultures; pouring plates x Bacterial transformation (chemically competent cells) Bacterial transformation (electro‐competent cells)

Harvesting cell cultures by centrifugation Use and transport of liquid nitrogen, including caution about glass x Bradford protein assay Spectroscopic determination of DNA concentration (Cary UV‐Vis) Spectroscopic determination of DNA concentration (Nano Drop)

Plasmid mini‐prep kit Shared lab items: The Biochemistry lab groups share several lab items apart from the equipment noted below, but it is the PI's responsibility to share these items, not the students'. This policy prevents loss of expensive or easily misplaced items (e.g., quartz cuvettes, etc), and it prevents accidental contamination of sterile glassware or sterile areas X that would ruin experiments. Items located in the main Biochem teaching lab are available for sharing. Items located within a particular research lab are not available for sharing. This includes simple items such as wash bottles and sharpie markers, because both of those items could cause contamination issues. If someone asks you to borrow Shared work areas: The Biochemistry lab groups share work space in the Biochemistry Teaching Lab (311) and Prep Room (311A), as well as the Autoclave Room and Cold Room. These rooms require special X vigilance on your part to keep them clean for all users. Please be courteous of your fellow biochemists and clean up after yourself Autoclave training, logbook use, calendar sign‐up, courtesy policy (i.e., do not take someone else's autoclave load out without telling them, do not put a load in when someone else is signed up on the calendar, X remove your own load promptly).

‐80 ultracold freezer training, including policy on keeping the door closed at all times (remove your box and look through it on the bench while the door is closed, then return the box to the ‐80; do not stand at the ‐80 with the door open while you search for items). The X temperature alarm goes off at temp > ‐70 deg C. Press mute to temporarily mute the alarm. If at any time the temperature is above ‐60 deg C notify faculty members (Forconi Fox Riggs‐Gelasco Rogers etc ) Sorvall centrifuge training, logbook use, clean‐up procedure (i.e., proper lid attachment, understanding max speeds for different rotors, proper balancing of all centrifuge bottles and tubes, thoroughly wiping out each rotor well after use and then wiping with damp paper towel, turning x centrifuge off while door is open to thaw and vent the refrigeration chamber, wiping refrigeration chamber once it has thawed) Eppendorf centrifuge training, clean‐up procedure (similar to above). Shaking incubator training, including max speeds, balancing the weight across the platform, and never inserting a container into a clamp that was not designed to hold that size and shape of container.

Instrumental/ MilliQ water system operation, including when to put it into Standby Equipment / mode (always return it to Standby mode, unless the tank is < 90% full. In Shared spaces X that case, wait for the tank to refill, then come back and put it in Standby mode ) PCR thermocycler operation, including reminder to press Enter before ending a reaction, so that the lid retracts (do not force the door to open if the lid has not retracted) and sign‐up calendar. UV/VIS

FPLC

Gel Doc training. This instrument in the Prep Room includes an area where ethidium bromide is frequently used. Gloves are mandatory, including on the laptop. Once you are done, throw away your gloves, wash you hands, and put on new gloves. Nano Drop training. This instrument in the Prep Room is a quick way to determine oligonucleotide concentrations. See Dr. Fox for training.