General Rules for Working in the Chapela Lab

General Rules for Working in the

Chapela Lab

The following are some general guidelines for our lab. There are many people sharing the workspace in here. Please be concerned for the health and safety of us all. Use common sense in working with all materials. If you are unsure about anything in the lab, please ask!

The most important aspect of life in the lab is, first, assuring the safety of yourself and your lab mates. The next is respecting the communal nature of the lab. Although there are not very many chemicals in our lab which pose a severe health hazard (there are some), there are many pieces of equipment and chemicals which are shared by and necessary to many of us in the lab. Proper use of both will ensure that our lab functions smoothly. If you are unsure how to operate any piece of equipment or you are uncertain about the use or disposal of chemicals, ask your elder lab mates!

I. General Cleanliness and Orderliness

1. Room designations. Room 208 is for clean cultures, Room 210 is for PCR items and Room 4 is the dirty lab. The rule of thumb is no "dirties" in any room but 4 and no PCR products in any room but 210 (except 208 hood). The hood in 208 has priority for PCR prep and 210 is always for microbiology.

2. Clean up after yourself. If you spill it, clean it up. Wipe off bench tops when finished working. Leaving things out overnight is fine when you are using them but make sure it isn’t forever.

3. Glass Ware. You are responsible for washing your own glassware. We keep a certain amount of common items in the lab. More is available in the stockroom in 148 Hilgard. The graduated cylinders and funnels are the only Chapela Lab owned items and should not be returned to 148.

4. Balances. The balance area should always be cleaned after use. Wipe the balances and around the balances with a damp towel. For the fine scale balance take the tray off and clean under the tray.Turn put balances in standby mode after use.

5. Labels. Label your bottles, flasks, etc. We don’t know if it is H₂0 or HCl.

6. Read labels of all items carefully. Things labeled PCR-use should only be used on PCR products. Things labeled Hood Use Only should not be removed from the hood, especially the hood pipettes. Pipettes have minimum and maximum points, read these as well. They are .5-10, 10-100 and 100-100uL. Don’t over or under set for they will break or become uncalibrated. One 10-100uL pipette was found set to 6.5uL. THIS WILL BREAK IT.

7. Pipette tips. Everyone should fill and autoclave their own ultramicro (.5-10uL) pipette tips, do not use ART or benchtop Enviro-tips as a backup. They are there for their own reasons. Shared tips (10-100 and 100-1000uL) should be filled whenever you use the last box or you notice they are low. Enviro-tips should be left on benchtops. ART tips should be replaced when there are less than a handful left in the box. Don’t leave it for the next person. Empty boxes should be placed in the cabinet in room 208 marked pipette tips on the right side of the top shelf. DO NOT leave empty boxes in the hoods or on the bench tops.

8. Eppendorf tubes. Fill up Eppendorf tube canisters and prepare for autoclaving when you use the last one. If there is not enough material to do an autoclave run, leave it with other items in the tray at the end of the benchtop in room 208 marked Autoclave Prep Area. It there is enough material and you are able to, autoclave it yourself. If you are running the autoclave check the prep area for materials to be autoclaved.

9. Laminar Flow Hoods. Clean up hood after each use. That means...

Room 4 Hood:

· Sterilize surface with ethanol.

· Place all inoculation tools back in the drawer below the hood.

· Put ethanol bottle back on top of hood.

Room 208 Hood:

· Pipettes should be paced back in holder and all regular hood items left neatly. Regular hood items include bunsen burners, inoculation tools, and eppendorf racks. All pipette tip boxes should be placed back on the benchtop to the right of the hood in room 208. Also, do not leave solutions in ice-packs or where they do not belong, place in your regular freezer area or back in stock solutions. IF THERE ARE ITEMS LEFT IN THE HOOD THAT BLOCK THE FLOW OF AIR THE HOOD SERVES NO PURPOSE.

· Sterilize surface with ethanol

· Papertowels, chem-wipes, and ethanol should be kept on top of the hood.

Room 210 Hood:

· Sterilize surface with ethanol when done

· Papertowels, chem-wipes, and ethanol should be kept on the edge of the hood.

10. Microscopes. Always cover microscopes after using them. Always clean oil off of high power lenses immediately after use. If you don’t know how to use them, please ask for help! These are high quality, expensive pieces of optic equipment. Treat them that way.

II. General Supplies

1. General supplies are stored throughout the lab. The cabinets and drawers are marked. Please familiarize yourself with where things are. When you notice that we are low on or out of some stock item, please write it down on the Items Needed in Lab sheet on the wall inside the door of Room 210. The Order Log located behind this clipboard lists the supplier of most items. Someone should be in charge of ordering.

2. Field Supplies. Some field supplies (bags ,tags, tools, equipment and more) are located downstairs in the locked cabinet in Room 4, See someone in the lab for the combination.

3. Chemicals are stored in both rooms 208 and 210. The bulk of the chemicals are in 210 in the glass cabinets in alphabetical order. There is a list of all of our chemicals on the door of Cabinet S-Z When you add a new chemical to the cabinet please note it on the back page of the Chemicals list.

III. PCR in lab

1. When you are on the last tube of Taq, make it your job to purchase more from the Wellman Courtyard storeroom. We should never be down to just 1 half-empty tube or a bunch of very empty tubes. Use one tube at a time, throw out when empty and then open a new one. If you aren’t able to go to the storeroom, write it down on the Items Needed in Lab list by the door in 210 and on the dry erase board in room 208.

2. There are common solutions for the lab that we rotate in making. They are PCR water, PCR TE, CTAB, General 1.5% Agarose, dNTP’s and PCR Buffer. If you notice one of these becoming low, look on the cover of the Recipe list folder to see whom is next. Recipes for all the above should be in the folder. The folder lives above the Thermal-Cycler. If there is a change in the recipe, please mark it down (eg- changing from TAE to TBE for general use)

3. Electrophoresis buffers should always be clear. Remember to use the same %solution as the gel you are running for best results. We use TBE in general, TAE for DNA retrieval (physical removal from the gel). Please mark on the bottle label when it has been used (a simple slash will do). For quick and dirty gels (checking for product) use an older bottle. When they become too dirty with muck, make new ones. Old buffer can be poured down the drain. Clean bottles and add the appropriate amounts of stock solution + water. Amounts are written on stock solution containers, located on the top shelf on the other side of the buffer bottles.

4. At times when there is a strong demand for the thermal-cycler (PTC) please use the sign-up sheet located above the thermal-cycler. It is also a good idea to sign up if you know that you will need to use it at a specific time, even when usage is light

5. Ethidium Bromide and UV Light Table in THE BOX. EtBr is a powerful mutagen (this should come as no surprise since it binds tightly to DNA at low concentrations). You should always wear gloves and a blue lab coat (located in room 210 beside fume hood) when you work in THE BOX. You should also consider everything in there to be contaminated. If you are splashed with EtBr, wash any exposed areas immediately and thoroughly. The good news about EtBr is that it is light sensitive, so lightly contaminated surfaces will not remain contaminated forever. Dispose of gloves and paper towels into white trash bin below imaging area. Dispose of gels in the bag in BOX. Always rinse illuminator with H2O after use.

6. If gels are starting to look faded, add more ethidium bromide to staining solution, 5-10 drops. If staining and de-staining solutions look very dirty, change them out by dumping solutions in glass jars under fume hood (careful, it is ethidium bromide) and then make fresh solutions. It’s just water or water + EtBr, to about the same level as usual. We don’t use exact measurements for this, so eyeball it.

IV. Equipment

Most of the equipment we have in the lab is fairly standard and not difficult to operate, but you should ask someone to show you how to operate a piece of equipment before using it for the first time, even if you have used similar items in the past. There is a drawer in each lab room that contains manuals for the equipment in that room. Some equipment (e.g. Lyophilizer) require routine maintenance; fill in user log for such equipment.

V. Disposal of Solid and Liquid Waste and Hazardous Materials

There are various methods of disposal of waste in the lab

1. The following need to be disposed of by EHS: When these bags are full, follow procedures in the Lab Safety Manual (located above PCR machine) for requesting pick-up

a. Gels go in a plastic bag in THE BOX

b. EtBr contaminated gloves and paper towels go in the white trash can directly below THE BOX

c. EtBr liquid waste goes into the container under the fume hood. Be very careful when transferring waste to here. Go slow, use the funnel, wear gloves. Leave the funnel in the dish.

d. Phenol, chloroform, etc. contaminated tips and tubes (lower phase from extractions) go in the waste bucket in the corner of the fume hood. Empty the contents of the tubes into the organic waste container in the hood.

2. Broken glass goes in the blue and white glass box in 208 under the sink.

3. Sharps go in containers (yellow and red) labeled as such in both rooms.

4. Old cultures – solid and liquid should be autoclaved before disposal. There are autoclave bags in drawer in 208. If you do not know how to use the autoclaves in the building, please have someone show you before using. They are old and finicky.

VI. Lab Safety Manual/Chemical Inventory

There is a lab safety manual that is located in the glass cabinet next to the freezer in room 210. There are detailed drain/chemical disposal guidelines, chemical inventory instructions and lab safety information. All Standard Operating Procedures (SOPs) are contained in this manual. Before starting any work in this lab you must read both The Lab Safety Manual, Chemical Hygiene Plan and then sign the Chemical Hygiene Plan.

VII. Permits and Inventory of Soils and Imported Biological Materials

Our lab has permits for the importation of fungi, soils, and plants from most countries. The original copies of these permits are located in room 210 in the binder marked "Permits and Imported Materials Inventory". If you are traveling abroad and need a permit make a copy of the permit required and obtain the required packing labels from the binder. DO NOT TAKE ORIGINAL PERMITS WITH YOU.

All imported materials must be inventoried. When you bring in new materials fill in the appropriate inventory log found in the binder marked "Permits and Imported Materials Inventory".