Version March 5 2025

 

Gelb Lab Policies

 

Careful reading of this enitire document is required of all Gelb lab members.  Read all sections even if you will not be involved with some of the items (i.e. radioactivity).    The policies listed in this document are to be stricktly adhered to unless you get specific permisson from Gelb to do something differently.  These policies are the result of years of experience and are necessary to make the lab run smoothly and to prevent loss of time and resources. 

 

 

 

1. Lab safety.

1.  Lab goggles/glasses must be worn by all workers at all times when working in the lab (even if you are doing mostly biological work).

2.  Gloves should be reused if they are not contaminated.  Just carefully remove them and place them on your bench for reuse.  Unless you are using highly toxic reagents, you should not have to use more than 2-3 pairs of gloves per day.

3.  Know where the eyewashes and fire extinguishers are located in the labs.

4.  If there is an emergency, dial 911 on campus phones.

5.  Water condenser houses should be fastened with copper wire, and water flow should be turned as low as possible for reactions overnight.  Use caution when pulling tubing off of glassware, if it is really stuck, use a razor blade to cut away the tubing.

6.  Never put bleach or oxidizing metals (i.e. Cr(VI) and Mn(VII) salts), hypervalent iodine reagents, H2O2, etcɠinto organic waste containers as they will react violently with organic material.  Never mix oxidizing acids like nitric acid and aqua regia with organics, they can react violently.  Metallic lithium should never be placed under a N2 atmosphere; this will lead to a violent reaction to form Li3N.

7.  Be careful with highly toxic reagents (thallium salts, alkyl mercury salts, tin reagents (especially tetraalkyl or trialkyl aryl Sn compounds), alkylating agents (MeI, dimethylsulfate, etc..).  Clean up any spills (in your hood, bench, balances, etc..) immediately using appropriate procedures and dispose contaminated items in a separate bottle in a hood. If you spill one of these items call UW Environmental Health & Safety to find out how to properly clean up the area.

8.  Be sure to keep your hood sash down as much as possible, especially when evacuating glass containers > 500 ml (they can implode).

9. YOU MUST READ THE SOP FOR HANDLING CHEMICAL WASTE ON THE UWCHEM WEB PAGE (SAFETY SECTION).

 

2. Lab Notebooks.

1.Of all the costly items which constitute a research lab, it is perhaps surprising that the most valuable is your research notebook. If properly kept, it is a complete record of scientific activities in which you no doubt have invested thousands (yes, thousands) of hours. The sound of a fire alarm has caused more than a few graduate students to grab their research notebooks before fleeing a potentially burning building. When it is time to carefully describe your scientific activities in a publication or your thesis, you will rapidly discover that these notebooks are indispensable: The human mind simply cannot remember every minute detail of so many experiments. Additionally, it is not uncommon to attempt to reinterpret experimental results years after the original observations were made. Without the notebooks, this would clearly be impossible. Without careful records, we are all doomed to be constantly repeating our work.

1. The lab purchases hard bound notebooks from a company, and these are the only notebooks that can be used.  Do not use water soluble ink or pencil.

2. Hard copies of all data must be placed in your notebook.  This includes chart recording output (i.e. HPLC traces) dried SDS-PAGE gels, photograhs, etc.  Tape or stable these into your notebook pages at the appropriate place.  Do not put them in separate binders.  The only exceptions are NMR and mass spectra because there are so many of these that it will make your notebook bow too much.  Put these in a 3-ring binder, in the proper order according to the date, and be sure each is labeled with the appropriate compound label name (see below).  Do not store original data on your personnel computer; put hard copies in your notebook.

3. Each bound notebook must have a table of contents which lists all the sections for the entire notebook. 

4. Each day that you work in the lab you must begin that page of your notebook by placing a date on the page.

5. You must record your observations within minutes of making them. If you are collecting recorder output data, you can jot down some specifics about the experiment (i.e. how much enzyme, inhibitor, chart speed, etc...) on the output, and you should assemble the data into your notebook within a day or two after the experiment. The details of the design and components of the experiment should be recorded in your notebook as you set-up the experiment.

6. All experiments, regardless of whether they "worked" or not, regardless of whether you are up or down about the results, must be recorded. Often the details of failures are the most informative.

7. Compounds must be properly labeled with a unique identifier.  A good system to use is your initials, then the date, and then a sequence number in case you have more than one label on a given day, i.e. MG-4-1-08-1.  This label name has to be unique.  The label name has to be on the container of your sample, on the various data items that link to that compound (i.e. NMR, MS, HPLC trace, etc..) and in your notebook.  Different batches of the same compound (made at different times or made at the same time but of different purify) need to get a unique label.

8.  You need to list the commercial sources of materials being used.  For example, benzoic acid (Aldrich), G25 Sephadex superfine (GE Biosciences), dry THF (gelb lab solvent drying system).  The sources of very common reagents (i.e. NaCl, sodium phosphate, KOH, etcɩ do not need to be listed.  If the company sells more than 1 kind of the same material (i.e. Chymotrypsin from Sigma) be sure to give the Catalog number.  For biomolecules (proteins, etcɩ you need to list the lot number.

9.  The level of detail to describe experiments needs to be such that someone trained in the area of synthetic chemistry or biochemistry, for example, would be able to reproduce your work with your notebook in hand.  So this means that you can say, for example, under N2, but you donմ have to draw a picture of the apparatus in your notebook.  If you dry a solvent, be sure to say how this was done (i.e. distilled from CaH2 under N2).

10.  For TLC plates, you donմ have to put them in your notebook, instead draw a picture of what the plate looks like and be sure to indicate the visualization method and solvent.

11.  All NMR should indicate the solvent and the standard that was used to assign the chemical shift scale.  Same for IR spectra.

12.  For column chromatography (silica, HPLC, gel filtration, etc.)  give the dimensions of the column, the packing material, the vendor, the flow rate, the volume of fractions collected, the solvent composition and other needed details (wavelength for HPLC detection, etcɩ.

13.  Give the temperature for all chemical reactions or biological purifications even if it is ambient temperature.  This includes organic reactions, enzymatic assays, biological purifications, etcɼo:p>

14.  If you receive samples from another lab (as part of a collaboration) the documentation that came with the samples must be placed in your notebook in the appropriate place.  If no documentation came with the samples please get it from the collaborator.  The collaborator must provide you with a specific identifier that they will be able to use to know later what samples they sent you.  If the collaborator does not supply with you with a specific identifier, you must request one from them.  It is not accepable to just say ҳamples received from lab X on March 1, 08Ӯ

 

3.  Water.

1.  Use house deionized water to prepare bacterial growth media and final rinsing of glassware.

2.  Use Milli-Q water to prepare all buffers and HPLC water-based solvents. 

3.  Do not store purified water since impurities will leach out of the containers.  For HPLC, it is best to prepare a fresh batch of filtered Milli-Q water before use.

4.  Store all buffers at 4 deg C since buffers left at room temperature can support microbial growth.

 

4.  Solvents.

1.  Always buy the large bottles of solvent (typically 1 gal or 4 liters) when possible.  Do not buy small bottles except for very odd solvents like dioxane.  Do not buy small cans of ether.  Some solvents are bought from the bulk containers in the stockroom by refilling a container from the Gelb lab (i.e tech. acetone, 95% ethanol, etc..). Be sure to check with the solvent price lab person to find out which ones. Solvents add up to a large amount of our supply bill so you must be wise in buying solvents.

2.  There are special solvents in the Gelb lab stockroom that are earmarked for use in the solvent drying system.  These are predried solvents and are more expensive than other solvents.  These solvents are to be used only for the solvent dryer system.

3.  Old bottles of ether including diethyl ether and THF are an explosion hazard, especially if allowed to evaporate to a small residual volume.  Avoid having these on your bench.  If you spot an old bottle of THF or other ether, report the bottle to the person in charge of ҥthersӮ

 

5. Chloroform.

Most commercial bottles of chloroform contain ethanol as a stabilizer to prevent photo-decomposition to give HCl and other species. The exception is bottles of deuterated chloroform for NMR (CDCl3). Over time, these bottles will develop HCl and other impurities that can react with your molecule. Thus, it is a good idea to add a layer of molecular sieves (about 0.5 cm high) to each new bottle of CDCl3. This tends to reduce the build up of reactive species. Old bottles of CDCl3 that do not have molecular sieves tend to develop a pungent order that is readily apparent when the bottle is opened.

 

6.  Plasticware vs glassware.

Beware the many organic solvents cannot be stored in plastic containers.  Polypropylene and polyethylene (low and high density) are compatible with weak and strong acids, weak and strong bases, alcohols, aldehydes, ketones, esters, but not with hydrocarbons, halogenated hydrocarbons, and strong oxidixing agents. Polyethylene is not autoclavable but polypropylene is. Most eppendorf tubes are polypropylene but check to be sure. Many plastic beakers and graduate cylinders are polypropylene but check to be sure. The trade name Nalgene does not necessarily imply polypropylene.  Polycarbonate and polystyrene are degraded by most non-aqueous solvents (if you don't believe this, squirt some acetone onto a piece of styrofoam).  Teflon (also called polytetrafluoroethylene or PTFE) is resistant to essentially all solvents.  Clear plastics (not frosty) are almost always not polypropyelene or polyethylene and most are degraded by most non-aqueous solvents.

 

7. Barcodes

 We are required by State Law to keep an accurate chemical inventory for chemicals in our research labs. Please follow the following rules:

 1. All incoming chemicals that you pick up from the Chemistry Department Stockroom that are barcoded by the stockroom must be laser scanned at the place in the lab where the item will be placed. The stockroom will barcode most items except for very small bottles.

2. When you discard a bottle with a barcode, you must laser scan the barcode in the delete mode.

3. If you take a barcoded chemical from one Gelb lab location to another, you must laser scan the item with the laser pen in the new location unless you return the chemical to its original location within the same day.

4.  If there is no laser scaner in the room where the barcoded item will be kept (i.e. tissue culture lab or cold room) use the laser scaner that is closest to the storage room.

 

8. UWCHEM Chemical Inventory.

When compounds are barcoded in the stockroom they are placed into a computer database.  It is possible to search this database if you have appropriate software loaded on your computer.  To do this ask one of the veterans in the lab or you can ask the computer support staff.  The use of this software is a great way to locate a chemical anywhere in UWCHEM in case you need to ask another lab for a small aliquot of a chemical.  In this way we can avoid always having to buy a large bottle of a chemical when we need a small aliquot.  It also saves time in that you donմ have to wait for the ordered chemical to arrive.  Be sure you learn how to use the software.  For example, it is best to surround the chemical name by the symbol %, i.e. %isobutyl%.  The % is the wild card string of any length.  This is important because stockroom folks may enter the chemical name into the database as 4-hexenoic acid or hexenoic acid; you get the idea.

 

9. Stockroom, ordering supplies and incoming packages

1. You are forbidden by UW/NIH policy to buy office supply items such as paper, rulers, pencils, staplers, pens, etc.  You are to provide these on your own.  Also there is an unlimited amount of scratch paper around the department if you need some.

2.  You can purchase tape (labeling tape and scotch tape) and Sharpie pens from the stockroom since these are considered to be research supplies rather than office supplies.

3. Disposable supplies such as pasteur pipets, disposable test tubes, Eppendorf tubes, Falcon tubes, gloves, weigh boats, solvents, salts, gas tanks, standard pH callibration buffers, etc.... may be purchased in the stockroom.

4.  Avoid buying non-disposable glassware and plasticware (i.e. Erlenmeyers, round bottom flasks, graduate cylinders, etc..) since we have a large supply in the lab.  If you are in need of more of these items, talk to Gelb.

5.  Try to buy the largest bottle possible for things like buffers, salts and solvents since the price is much cheaper.  We will certainly finish up a large bottle of such items in time.  For solvents, be sure to talk to the Gelb lab person in charge of the ғolvent PricesӠlab job.  This person can tell you what kinds of solvents to buy to save money. Solvents add up to a large amount of our supply bill so you must be wise in buying solvents.

6.  If you pick up a package for Gelb or other lab members, be sure to notify the person that the package has arrived and if the person is not arround, be sure to store the item in the appropriate place (check the label on each item to be sure how to properly store the item, i.e. 4 deg C, -20 deg C, -80 deg C, and room temp.) and to leave a note on the perrsonճ desk where the item is.

7. All order forms must be signed by Gelb or the Senior Research Technician/Scientist. Before ordering chemicals, be sure to check the Gelb Lab to make sure we don't already have it. Also if you need a small amount of a chemical, check the UWCHEM Inventory  since you may be able to get a small aliquot from another lab. You may be asked by the person signing the order form to check other sources for a better price when appropriate. When ordering from commonly used vendors (Aldrich, Sigma, Pharmacia, Avanti, etc....) ask other lab members if they need anything from these companies; combining orders will save shipping charges. If you notice we are running low on an item that will certainly be needed in the future (TLC plates, scintillation fluid, Tris, etc....) be sure to order the item well in advance since it may take 1-2 weeks to receive it. Good bargains that you become aware of should be announced at the beginning of group meetings.

8. Aldrich may be more expensive and charge more for shipping chemicals. Before ordering from Aldrich check the online VWR and Fisher catalogs as they carry chemicals offered by Agros and TCI. If they dont have the item, check the TCI America, the Agros catalog and the Alfa/Aesar catalog. Order from Aldrich are allowed only if these other vendors don't have the chemical. Also these other companies charge less for shipping than Aldrich does and offer a discount for UW customers so when you place the order write on the order form to ask the purchaser in the UWCHEM office to ask for the UW discount on the price of the chemical and on the shipping.

9.  It is important that when you order something that the P&A places the order with the vendor the same day that you give them the order form. Often they will have stacks of orders. Almost always they will order the item the same day if you ask them to, otherwise they often order the item 1 day later. The head of the P&A office has instructed the person placing the orders to order the same day if the person placing the order specifies this so donմ be shy to ask. If you are very much in a hurry for something you can check back with the P&A office the next day to be sure the order was placed. Don't wait a week to find out your ordered item has not arrived because it was ordered days after it was given to the P&A office. Also you should specify overnight delivery only in critical cases because it is considerably more expensive.

 

10. Chemical waste. 

You cannot store chemical liquid waste in your hood for any length of time.  When you wish to discard chemical liquid waste, pour it into the dedicated large plastic waste jugs. Ask the person in charge of chemical waste where these jugs are and what kind of waste can be placed in each jug.   We have had a few past incidences of a glass bottle of chemical waste break in a hood and it causes serious damage to the lab and is a serious health hazard.

 

11.  Flammables and chemicals in the lab.

You are not allowed to store 1 gallon bottles of solvents in your cabinets; they all must be kept in the dedicated solvent cabinets (yellow safety cabinets in the labs and chemical stockroom solvent cabinet). This includes the flammable solvents for all HPLCs. If you need to hook up a large bottle of solvent to the HPLC, when done with the HPLC remove the bottle and return it to the solvent cabinet, and place the solvent line in a small bottle of the same solvent, wrap the top of the bottle with foil to prevent solvent evaporation.  By storing the solvent line in a small bottle of solvent you will not need to prime the line once you return the line to the large solvent bottle. You can keep small (~1 pint) bottles or squirt bottles of solvents on your bench or shelves but be sure they are capped.  Do not store racks of test tubes full of solvent on your bench, put them in your hood.  The same applies for flasks or other containers containing solvents.

 

12.  Gas Cylinders.

We pay a rental fee per month on each gas tank that we hold in the lab. Thus, when you have an empty gas tank, please return it to the stock room as soon as it becomes empty. Be sure to return it with the metal valve cap.

 

13.  Cleaning up around equipment areas.

When you are done using a piece of equipment you should remove the items that you brought to the area (racks of tubes for HPLC, Pasteur pipettes, pipet tips, scintillation vials on top of the counter, etc...) and clean up any spills. Items left for more than a day or two in common equipment areas can be thrown out with prior notice. Also be sure to clean up the area in the cold room that you have used, especially buffer spills since these are corrosive.

 

14.  Disposing sharp items.

All disposable glassware and broken glassware that is beyond repair must be discarded in a cardboard box designated for such a purpose. Glassware coated with pungent or volatile toxic compounds should be cleaned in the hood before disposing in the sharps box. When full, tape the box shut and put it in the designated area for pick-up by the janitor.

 

15. Pipetting liquids.

1. The Gilson Pipetmen and similar devices use polypropylene plastic tips. These can be safely used with aqueous solutions, ethanol, methanol, and many organic solvents (Check a catalog such as Cole-Palmer for a Plastics Chemical Compatibility Chart). Do not use these devices for corrosive agents such as concentrated HCl, concentrated ammonium hydroxide, and strong acids that are volatile (i.e. trifluoroacetic acid) since the vapors will corride the pipettor. You can use these for concentrated weak acids such as acetic acid and formic acid or strong but non-volatile acids such as phosphoric acid and sulfuric acid.  Use glass graduated pipets to deliver corrosive and volatile liquids (i.e. concentrated HCl, trifluoroacetic acid, concentrated ammonium hydroxide).

2.  The person in charge of pipetman callibration needs to check the callibration of your pipetman every 6 months.   Do not use a pipettman that has not been callibrated in the past 6 months.

3.  Hamilton syringes can be used for all liquids except strong acids since these will degrade the metal plunger and needle. Also, never dry Hamilton syringes in the oven since this may melt the glue that holds the needle in place. Also, clean these syringes shortly after use so that they do not get jammed. Also never try to force the plunger into the syringe since this will likely put a kink in the plunger (especially those for the 10 ul syringes) and this will permanently damage the syringe.

4.  Delivery of small volumes of organic solvent (less than about 50 ul) using a pipettman is not accurate.  If you care about the accuracy of the volume delivered use a Hamilton syringe.

 

16.  Cold room use.

1. Nothing is to be stored on the floor. Keeping the floor clear will allow the janitor to periodically wet mop the floor. Items left on the floor will be discarded without notice.

2. No ringstands are allowed in the cold room. Rings stands rust, and rust creates mold. If you need to set up an item that needs a ringstand, use the aluminum lattice rod instead. No metal cans of any kind are allowed in the cold room (such as cans of chemicals from Aldrich, etc...) unless they are placed in a plastic tray. Avoid leaving metal items in the cold room such as binder clips, dustpans, etc... Stainless steel and aluminum items are OK.

3. No cardboard is allowed in the cold room (exception is boxes of film). Mold easily grows on cardboard. This includes boxes of tissue culture media, boxes of test tubes, etc....

4. Personal items must be stored in your designated area on the shelf, and the shelf must be labeled with your name. Piles of lose items such as Eppendorf tubes, Falcon tubes, etc... will be discarded without notice. Place loose items in a Tupperware container. Racks of tubes must be covered with foil and labeled with your name. All bottles must be capped (not left open or closed with parafilm). Uncapped bottles will be discarded without notice.

5. Tubes should not be left in fraction collectors unless the collector is actually in use. Tubes left in fraction collector racks more than 1 week will be discarded without notice.

6. Items should not be stored on the bench or on tables and carts. They should be discarded or stored on the shelf in your designated area. Items left on the bench, tables, or carts will be discarded without notice. The only exception are bottles of buffers next to the FPLC, but open bottles will be discarded without notice unless they are actually being used in an ongoing FPLC experiment.

7. No bare wood is allowed in the coldroom since mold grows quickly on exposed wood.

8. Puddles of liquid on the bench and on stirrers should be wiped up before and after your experiment. To insure that benches and stirrers are periodically wiped clean, experiments in progress on a bench or stirrer that are covered with liquid will be terminated and discarded without notice unless the spill is a result of a leak from the experiment such as a column that is leaking or a fraction collector that is stuck. This includes all surfaces of the FPLC.

9. Buckets of ice that are not actually in use will be discarded without notice. Buckets of ice that have been left to melt will be discarded without notice along with floating samples.

10. All centrifuge rotors are to be stored only in the designated area, not in the cold room.

11. The cold room is mainly for biological work. Organic reactions that need to be stirred in the cold for long periods must be kept in tightly-capped vessels with parafilm wrapped around the cap/vessel so that vapor escape is kept to a minimum.

 

17.  Equipment use by other labs and given out reagents.

1.  All requests by outside Gelb lab members to use Gelb lab equipment must be cleared by Gelb.  If Gelb is out of town, the person can ask him by email or wait until he returns.

2.  People outside of the Gelb lab can have small aliquots of reagents as long as the estimated cost is < $5 and the person is part of UWCHEM.  Requests from other departments need to be approved by Gelb.

 

18. Balances.

 After you use the balance, inspect the balance and the area around the balance for spills and wipe the area(s) clean. Don't just brush the spilled chemical off the pan and on to the table. Turn the balance off when done and leave nothing on the pan.

 

19.  Refrigerators/Freezers.

1.  Bottles of flammable or volatile chemicals (i.e. TMS-chloride, n-butyl lithium in THF, etc) and most reagents for organic synthesis that require low temperature storage must be stored in the dedicated explosion-proof refrigerator/freezer units. 

2.  Items that should be desiccated should be in tightly-closed jars containing active desiccant. The jars should be labeled (i.e. frozen chemicals A-G).

3.  Small vials of your solutions in flammable solvents or your neat chemicals can be stored in non-explosion-proof refrigeration/freezer units as long as they are in tightly-closed jars.

4.  The freezer for storage of radiochemicals in the radioactivity synthesis lab must be locked when not in use.

5.  If you notice a cooling unit is not at proper temperature, report the problem immediately to a lab member who is in charge of freezers and refrigerators. If the person cannot be found, call physical plant. They can be reached 24 hrs a day in an emergency.

If a unit needs to be emptied (for repair or defrosting), be sure to temporarily store the items in an equivalent unit (do not put freezer items in a refrigerator, put them in a freezer).

6.  Each lab member will be assigned an area in a refrigerator, -20 and -80 freezers. Your name must appear on the unit door indicating the location of your area.

 

20.  Vacuum Pumps

Many of you will be given a vacuum pump that will be yours to use. The vacuum pump must be kept in a specialized cabinet designed for such equipment, and the pump exhaust must be connected to the exhaust manifold inside the cabinet. You should change the pump oil on a regular basis (3-4 times per year, more often if necessary), and you must indicate the date of the last oil change on your pump. There should be a cold trap in the vacuum line, and the trap should be filled with coolant before the pump is turned on; the trap should be routinely emptied. Usually you donմ need to use a cold trap if you are removing trace amounts of solvent. Avoid drawing strong acids into the pump (HCl, trifluoroacetic acid, etc) since these will damage the metal parts. If you have these acids in your sample, you must put a cartridge of NaOH or KOH pellets in the pump line. The pump must sit on a metal tray to collect oil. Although the machine shop provides a convenient and rapid repair center for pumps, it typically cost $200-$500 to have a pump repaired.

 

21.  Locking Lab Doors.

When you leave in the evening, be sure to see if others are still in lab. If not, check all lab doors to be sure they are locked (not just shut but locked). If you are working in the evening (after 7 pm) be sure to leave the doors locked on all labs except those that you are constantly using. Labs that you are not using or are using infrequently should be left locked. Lab doors should be locked before group activities such as journal club and group meeting.

 

22.  Hydrogen Halide Forming Substances.

Compounds which generate hydrogen halides when exposed to water must be stored at all times in designated glass bottles with teflon-lined screw caps. There is only one cabinet in the stockroom where HX formers can be stored. Glass bottles must be labeled with label maker with chemical-resistant label tape. Compounds which generate hydrogen halides include: PCl3, PBr3, POCl3, PCl5, SOCl2, oxalyl chloride, acid chlorides and bromides, chlorocarbonates (chloroformates, carbobenzoxychlorides and bromides), etc... If you are not sure whether a compound is potentially hydrogen-halide generating, it is your responsibility to find out either from Gelb or another reliable source of information. Aqueous solutions of HX can be stored in the Acids Cabinet.

 

23.  Centrifuge Rotors.

After using a rotor, inspect it carefully, and if there is any sign of spilled buffers, rinse the rotor well with water to remove traces of salts since salts will lead to rotor corrosion.

 

24 .Tissue Culture Lab.

1.  Use of the tissue culture lab requires authorization by Gelb. All tissue culture supplies must be kept in tissue culture lab, do not mix them with supplies not used for tissue culture.

2.  People outside of the Gelb lab cannot use our tissue culture lab.

 

25.  Broken Equipment. 

If you find that a piece of equipment is broken, you must initiate the process of getting it repaired. Do not return it to its storage location without taking further action. Broken major equipment items should be reported to the lab member assigned to that piece of equipment and they should take the necessary steps to get it repaired. For small equipment items consult with Gelb before getting it fixed in the shops unless he is out of town and then use your best judgment. Of course if the item can be easily repaired (for example a new fuse on a variac) make the repair yourself.

 

26.  Shop usage.

Requesting services from the machine shop or electronics shop requires permission from Gelb except for the repair of vacuum pumps.

 

27.  Silica gel.

Although silica gel may appear to be harmless, it is actually quite a health hazard, especially the fine mesh type for flash chromatography, which easily becomes airborne. It becomes permanently dislodged in lung tissue. Thus, all manipulations with silica gel are to be performed in your hood (opening of containers of silica, loading and running flash columns, etc...). Used silica is to be stored in dedicated container that is tightly covered.

 

28. Cuvettes.

1. Some cuvettes are made of glass and some of quartz. if you are working in the UV range (less than about 350 nm) you must use a quartz cuvette because glass absorbs UV light.

2.  Cuvettes are used by many different researchers. We have found on several occasions that some sort of oxidant can build up on the walls of the cuvette which dramatically interferes with the Ellman assay for SH groups. The Ellman thiolate that forms in the assay can rapidly reoxidize back to the colorless Ellman disulfide; this leads to a gross underestimation of the amount of SH in your sample, especially when small amounts of SH are being detected. The problem can be solved by soaking the cuvette in concentrated nitric acid for 10-15 min. followed by washing well with water. Then calibrate your Ellman assay with small amounts of standard cysteine to make sure that the yellow signal is stable and that the change in OD is close to that expected using the known extinction coefficient for the Ellman thiolate. You should carry out these steps each time you do an Ellman assay.

 

29. HPLC

1. Salts can only be used on a biocompatible HPLC.  If you are not sure, ask since salts will ruin a non biocompatible HPLC.  If you use salts, you MUST rinse the solvent lines and sample look and inject port with salt free solvent otherwise salts will precipitate in the system when non-aqueous solvents are introduced later.

2. Retention times on a 2 pump system are more reproducible than those on a single pump machine.  The single pump machine mixes the solvent via solenoid values prior to the pump head (low pressure mixing) and is not as reproducible in generating solvent compositons as a 2 pump system (high pressure mixing).   If you are doing prep work, a low pressure mixing is fine.

3.   Any water solvent used on the HPLC must be Milli-Q.  Organic solvents must by HPLC grade.  All solvents must be filtered through the Nylon-66 membrane filter.

4. All HPLCs must have an inlet filter installed, never remove this filter unit. The person in charge of the HPLC is responsible for changing this filter when clogged.

5. Never exceed the maximum flow rate recommended for each HPLC column by the manufacturer.

6. When you start it is important to be sure the column is clean. To clean, run a short gradient from 100% solvent A (more polar solvent) to 100% solvent B (less polar solvent), and continue to wash with 100% solvent B until no more peaks are detected or at least 5 column volumes has passed through the column. Washing steps can be done at a higher flow rate to save time as long as you don't exceed the maximum recommended flow rate.

7. Before injecting your sample be sure to fully equilibrate the column with initial solvent by washing with at least 5 column volumes otherwise your runs will not be reproducible. Be sure the loop is flushed with starting solvent (this is accomplished by leaving the injector in the inject position during column equilibration) otherwise solvent B in the loop may drive your compound off the column very early. Be sure the injector is in the load position before injecting your sample. If it is in the inject position the sample will exit one of the injector ports and come out on the bench or beaker depending on where the exit of this port is positioned. Never inject a sample volume that is more than 3/4 of the volume of the loop. The leading edge of the sample stream is pointed and will overflow the loop if you inject 100% of the loop volume. Never inject particulate into the HPLC, if you sample has particulate you must remove it by centrifugation or filtration before injection onto the HPLC. Also never inject a sample that has a pH higher than 8 onto the HPLC, alkaline solutions can destroy silica based normal phase and reverse phase columns and almost all of our columns are silica based.

8. When you are done using a reverse phase column, wash it with at least 5 column volumes of 100% methanol without TFA (methanol is perfered over acetonitrile since it is cheaper), and store it in this solvent. For column washing for storage be sure to have a flow rate zero step at the end of your solvent program otherwise you may forget to turn off the pumps, and pumping air through the column will often ruin it. If you remove the column from the machine, be sure to attach the screw-in end plugs so that the column does not dry out. To minimize lamp use, turn the detector on a bit before sample injection, and let it run for 5-10 min. to make sure the baseline is low. Turn off the detector when your are washing the column for storage.

 9. When you are running a compound for the first time it is important to run a solvent gradient covering extreme conditions (i.e. 100% solvent A, which is often water/TFA to 100% solvent B, which is often 100% methanol/TFA or 100% acetonitrile/TFA) and to continue to wash with 100% solvent B until no more peaks are eluting. After this initial trial you can fine tune your solvent program to save time. If you need to do a large number of repetitive HPLC runs (i.e. enzyme assays) it saves considerable time to find an isocratic (non-gradient) solvent since gradient runs require extra time to re-equilibrate the column back to initial conditions.

10. If you are making your first injection or if you don't want any cross contamination between your injections, i.e. if you are injecting different samples, you should flush the injection system to remove sample that may have been left from the previous injection. To do this put the injector in the inject position during column equilibrium so that the loop is flushed with fresh solvent. This will not rinse the injection port which often contains a pocket of liquid. To do this, turn the injector to the load position. We have a special plastic tip that attaches to a plastic syringe and fits snug against the injection port. Fill the syringe with flush solvent and flush the injection port with about 2-3 mL of solvent, the solvent will enter the loop and then exit the loop overflow line. You should also flush the injection system in this way if you do want cross contamination between samples.

11. If you are using the HPLC for radioactive compounds for the first time, you must discuss with Gelb how to do this and how to clean the HPLC column and pumps after use.

12. People outside the Gelb lab are not allowed to use our HPLCs or HPLC columns unless they have explicit permission from Gelb.

 

30.  FPLC

1. Before using the FPLC, you must get trained by the lab member who is responsible for the instrument and you must carefully read the Pharmacia FPLC manual before using the FPLC.

2. FPLC columns are nearly $1000 each so column maintenance is critical. Be sure to read the instruction sheet that comes with each column before using the column. Never store ion-exchange columns (i.e. Mono S and Mono Q) in sodium azide, store them as recommended by Pharmacia, usually in 24% aqueous ethanol. Gel filtration columns can be stored in aqueous sodium azide, again consult the Pharmacia instruction sheet.

When done with the FPLC, wash the pumps first with pure water, then with column storage solvent and then wash the column with this solvent. Then wash the pumps finally with pure water and leave them in pure water until the instrument is used again.

3. Always use milli-Q water to prepare solvents and filter all solvents through the Nylon-66 discs and be sure not to contaminate the non-buffer glassware with buffer salts. 24% ethanol in water can be filtered once and stored in the cold room in a plastic bottle. Milli-Q water for washing the pumps can be filtered once and stored in the cold room in a plastic bottle. Never store your buffer solutions at RT as microorganisms will begin to grow in them.

4. If the pump seals begin to leak, ask the person in charge of the FPLC for help in changing them.

5. Never inject a cloudy suspension onto the FPLC. All injected samples must be perfectly clear and they should be microfuged before injection to bring down any particulate. Particles can destroy the column.

6. Periodically it may be necessary to put a column through a series of washings with methanol, TFA, etc... as described in the Pharmacia manual. Ask the person in charge of the FPLC for help with this operation.

7. When a column is detached from the system always make sure the ends of the solvent lines are capped tightly to prevent drying out of the column.

8.  Use of Gelb lab FPLC columns by other labs is not allowed.

 

31. Stock solutions in organic solvents. 

When dealing with stock solutions of compounds in organic solvents, i.e. inhibitors, lipid standards, etc..  note that some organic solvents are prone to evaporative loss even if the solution is in a teflon-septum capped glass vial in the freezer.  For stock solutions where you care about the concentration of the dissolved component, you must do the following:

1.  Avoid containers where the meniscus is close to the bottom of the vial.  If the commercial stock solution comes with the meniscus close to the bottom of the vial, remove the solution from the vial with a Hamilton syringe (thus measuring the volume accurately) and dilute it with a known volume of the same solvent, and of course be sure to relabel the vial to indicate the new concentration.  If the meniscus is close to the bottom of the vial, it will be hard to mark the meniscus position (see below).

2.  You must mark the meniscus position on the outside of the vial.  Do not use a Sharpie since the line will easily rub off in time.  Use the label maker machine to print an arrow and place the label on the vial so that the arrow points to the meniscus.  In some cases you will have to reposition the commercial label so that the meniscus can be seen through the wall of the vial.  Do not mark the label with a Sharpie, rather print an arrow with the label machine.  If you remove enough solution such that the meniscus moves you need of course to reposition the arrow label.  Only by marking the meniscus can you be sure if there has been solvent loss due to evaporation (do not trust the vial cap to hold the solvent, experience has shown this to be unreliable).  We donմ want to repurchase expensive items (i.e. eicosanoids,etcɩ because the vial has gone dry and we have no easy way to determine the amount of material left in the vial.

3.  You donմ need to do this when the solvent is water or DMSO.  These will remain frozen when the vial is placed in the freezer, and evaporative loss will be minimal.  Other solvents need to be handled as indicated above, and it is best if you store the solution at -80 deg C to minimize evaporation of solvent.  Chloroform is especially bad in that evaporation may occur even if the vial is capped with a teflon-septum cap and the vial is stored at -80 deg C. Try to avoid chloroform if possible, but if you must use it for solubility reasons, be sure to keep your eye on the meniscus.  Never use methylene chloride or diethylether; they are simply too volative.

 

32.  Preparing stock solutions of accurate concentration.

1.  Stock solutions (i.e. internal standards, inhibitors, etc..) made to accurate concentration require special attention.

2.  Never try to weigh less than 1 mg of compound.  If you need to weigh 1-3 mg of compound, use the balance in Niels Andersonճ lab, which is accurate to 10 ug and be sure it has been callibrated in the past 2 months, otherwise callibrate it.  Be sure all solvent is removed by weighing the vial before and after the sample has been placed under vacuum (oil pump) 2-3 times to be sure the sample has reached constant weight.  While handling the vial, use forceps and wipe all the outside surfaces with a Kimwipe to remove fingerprints before you weight the sample.  Compounds of 3 mg or more can be weighed on the analytical balance in the Gelb lab; again be sure all solvent has been removed.

3.  If you have less than 1 mg, you can dissolve the compound in an accurately measured volume of deuterated NMR solvent, add a known amount of internal standard (using a Hamilton syringe) and use an amount of internal standard comparable in moles to the amount of compound you have.  Take an NMR using a recycle delay (time between scans) of at least 8 seconds.  Determine the amount of your compound by careful intergration of peaks from your compound and from the internal standard.  Calculate the moles of your compound from the relative NMR peak areas, of course correcting for differences in number of proton per NMR peak that is integrated.    Be sure there is no signifcant evaporative loss of NMR solvent.  Be sure to choose an internal standard that gives an NMR peak(s) that are well isolated from the NMR peaks from your compound and from the peaks due to the solvent.  Typical internal standards are DMSO (avoid methylene chloride, it is too volatile), CH3CN, etcɼspan style="mso-spacerun: yes">  (usually compounds that give singlets).

4.  For people working on the anti-parasite med chem project, use the SOP for preparing accurate concentration inhibitor stock solutions (see Gelb if you donմ know how to access this SOP).

 

33.  Radioactivity use

1.  Anyone in the lab using any type of radioisotopes must first take the UW Radiation Safety Training Course, please contact Radiation Safety to sign up for the next available course.  You must take this course even if you took a similar course before coming to UW.

2. All radiolabelled syntheses in which more than 50 mCi of material are used must be conducted in the hot lab. Glassware and other contaminated items are left in the hot-lab hood in a container filled with diluted "counts-off", then discard the spent liquid in the sink. You must clean up soon after you have finished (within hrs). When your done with the synthesis, leave nothing in the hood.

3. Store radioactive materials in the Gelb hot-lab freezer. Compounds must be in well-closed containers (leak-proof containers: either as supplied by the manufacturer or in teflon septum-lined screw capped vials) and labeled with your name, compound name, radioisotope, and approx. amount of radioactivity. It may be preferable to store certain items in the -80C freezer. This is allowed as long as the compound is not volatile (example that is OK for -80C freezer: 3H-farnesyl pyrophosphate, etc..; not OK are 3H-acetic anhydride, tritiated water, etc....).  See section 4 ғtock solutions in organic solvents.Ӽo:p>

 4. You can use radioactive material on your bench in a small well-defined area (usually marked off with taped down absorbant paper). However, if the material is at all volatile it must be used in your hood. Sometimes you will be leaving radioactive samples such as column fractions on your bench or in your hood. This is OK as long as they are in your designated work space for radioactivity and they are labeled as radioactive. High energy emitters such as 32P must be in shielded containers such as a plexiglass box.

5. If you are counting samples that contain more than 1 mCi of high energy emitters (32P, 35S, etc..) you must put a notice on the scintillation counter telling people not to open the door. Shortly after the counting is done you must remove your samples. If it is late at night and you want to count through the night, that's fine as long as you remove them in the morning shortly after the counting is done.

 

6. Radioactive waste should be disposed of shortly after you finish your experiments. Do not leave it on your bench unattended for any period of time. Dispose of waste properly as follows. You should almost always use Bio-degradable scintillation fluid that is water soluble. The contents of scintillation vials that contain < 50,000 cpm can be poured down the sink, and the empty vials can be discarded in the trash can (plastic vials) or in cardboard sharps box (glass scintillation vials, which are almost never used) after you have rinsed the vials with water. Other items of < 50,000 cpm can be poured down the sink as long as the item is not a hazardess chemical (certain organic solvents, etc...). After you pour radioactive waste down the sink, rinse the sink well. Scintillation vials and other containers that contain > 50,000 cpm are to be put in the radioactive solid waste bottle after their contents have been put in the radioactive liquid waste bottle. Solid 3H/14C waste is placed in the solid waste container in the hood in the equipment lab. Same for liquid 3H/14C waste.  The lab member that has been assigned the radioactivity lab job is responsible for maintaining the solid and liquid waste containers and having Radiation Safety pick up full containers. Solid waste of other isotopes are to be stored in your hood, again don't mix isotopes other than 14C + 3H.  Gloves can be thrown in the regular trash can as long as you have not spilled a significant amount of radiolabeled material on them, if so put them in the appropriate solids waste box.  Same goes for other solid items and remember liquid items of < 50,000 cpm can be poured down the sink unless the chemical is not disposable in this manner.

7. When you order radioactive materials you must put the room number where the item will be used on the order form otherwise the order cannot be processed.

8. If you run a gel of a protein labeled with 32P-ATP try not to run the dye front off the bottom of the gel. Then you can simply cut the bottom of the gel off with a razor blade and put this slice of gel in the 32P solids waste container. In this way you have safely disposed of > 99% of the cpm. The gel tank buffer can then be poured down the sink.

9. You must obtain special permission from Gelb to use radioisotopes other than 3H, 14C, 32P, or 35S. At such time we will discuss procedures for use and disposal of such isotopes.

10. This list of policies is a supplement to those from Radiation Safety and you are expected to proceed according to the requirements set by them.

11.  When using high levels of 32P (uCi to mCi amounts), it is inevitable that things like microfuges will get some contamination, even if you are very careful not to cause a spill.  Thus every time you use 32P, you must check the work areas with the geiger counter (centrifuges, bench, etc...), and if the counter registers radioactivity above the background, you must wash the relevant area with soap and water (wear glovers) until no signal above background is detected (or until the signal is very close to background).  The person handling the 32P is responsible for doing these geiger counter checks, and it must be done each time you handle 32P.  Please note that the lab member assigned to the radioactivity lab job will periodically monitor lab areas with the geiger counter, and  given that only 1-2 people in the lab are using 32P at any given time, it will usually be obvious who is responsible for a contamination that was not cleaned up.  Also please consider that large amounts of 32P left in the work area constitutes a safety hazard for everyone in the lab. 

 

34.  Decomposition of Radiochemicals.

Radiochemicals of high specific activity should always be stored in solution, not neat! When in solution, the emitted particles give up energy to the solvent. When stored neat, the emitted particles can lead to breakage of covalent bonds of the radiochemical. It is not uncommon for a tritiated compound of high specific activity (1-100 Ci/mmol) to decompose after a few weeks, even when stored at low temperature. The same is true for 14C compounds of high specific activity, approaching 50 Ci/mol.

 

35.  Storing Antisera

1.  There are only 3 types of aliquots of antisera allowed in the Gelb lab:

a.  6-12 ml frozen aliquots in 12 ml capped Falcon tubes.

b. 200 ul aliquots in 0.5 ml capped Eppendorf tubes (do not use 1.5 ml Eppendorf tubes!)

c.  10 ul aliquots in 0.5 ml capped Eppendorf tubes (do not use 1.5 ml Eppendorf tubes!)

2.  The 6-12 ml aliquots are stored at -20, the 200 ul and 10 ul aliquots must be stored in freezor boxes at -80 deg C (not -20  deg C).  Frozen aliquots will sublime at -20 deg C leading to drying out of the aliquot (especially for the 10 ul aliquots). This is not a problem for the 6-12 ml aliquots so they can be stored at -20.

3.  Never remove the freezer box from the -80  deg C freezer, open the box with it still inside the freezer compartment and remove the aliquot quickly.  10 ul aliquots will thaw in < 1 min if you remove the box.  It is important to minimize the number of freeze-thaw cycles since some antibodies are denatured after multiple freeze-thaw cycles.

4.  Remember polyclonal antisera cannot be replaced, so we must ensure proper storage and handling. 

 

36.  Base bath for synthetic glassware

1.  If you are doing mostly organic synthesis work, you should have your own base bath for washing glassware.  This consists of a plastic tank with plastic lid filled with isopropanol (use the bulk grade from the stockroom) and  enough KOH pellets to saturate the solution (visible pellets left).  Keep your base bath in the cabinet under your sink and covered with the lid.

2.   The base bath serves to remove the last layer of organic residue from your glass reaction vessels and also neutralizes any acidic sites on the glassware.  The latter can readily react with water sensitive reagents (i.e. organolithiums, Grignards, etcɩ especially when you are working on a small scale.  Thus, all glassware for use with water sensitive reagents should be soaked in your base bath for at least 3-4 hrs (overnight is convenient).  Remove the glassware from the base bath, rinse with with tap water, then with house deionized water and then with tech. acetone.  Dry the glassware in the usual way for working with water sensitive reagents (see handout in the synthesis section of the Gelb lab handouts).  Avoid putting heavily coated glassware in your base bath; remove most of the residue first.  In this way your base bath should last at least a few years (it is expensive to refill with new isopropanol on a regular basis). 

3.  If you donմ do synthesis on a regular basis, ask a synthetic chemist to use their base bath if you need it.

37.  Broken Glassware.

Glassware that is repairable (i.e. star cracks, broken joints, etcɩ should be given to the Gelb lab person in charge of glass repair, and this person will take it to the glass shop twice per year to get it fixed.  Donմ throw away glassware that can be repaired.