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General comments:  Please feel free to come to me AT ANY TIME with questions or help.  I am also happy to do any experimental procedures for you while you are away from the lab for any reason.  Please freely share equipment and chemicals with other members of this laboratory, and with members of any other laboratory.  If equipment is borrowed by other laboratories, please note down the name and phone number of that lab.  Plasmids, radioactivity, and mammalian cell lines do not leave this laboratoy without my prior consent.

Laboratory Notebook:

               The lab notebook is one of the most important facets of laboratory work.  The organization and timeliness of its contents is a reflection of the quality of your work.

1.      I will provide you with the specific notebook.  The pages of the notebook are prenumbered and in carbon copy duplicate.  The duplicate sheets of the notebook are for your records when you leave the laboratory; the original sheets and notebook are the property of the university.

2.      DO NOT write on the back side of any notebook sheet—this is not considered proper notebook etiquette.

3.      Set aside the first 3 pages of the notebook for an ongoing Table of Contents.  The table of contents is to be sufficietly detailed that anyone not familiar with the contents of the book can easily find the page(s) corresponding to a specific experiment.

4.      Each set of experiments is to be prefaced with a “Purpose” statement, followed by a general “Approach” (which may be described in pictures).  The following is an example:

Purpose:  EMSA study to investigate the binding of Pdx1 to the insulin promoter A3/4 element.

Approach:  Pdx1 will be transcribed in vitro using the TNT-T7 kit from Promega and the vector pBAT11.Pdx1, and will be used in an EMSA with a 32P-labeled probe corresponding to the A3/4 element.

5.      Pictures of gels, animals, cells, etc. should be taken in duplicate, if you desire a copy for your own duplicate pages.  Otherwise all pictures must remain in the original (white) pages of the notebook.

6.      Any new protocol developed by you and of potential use to other members of the laboratory should be written up, much as a cookbook recipe, and submitted to me as an MS Word file.  This file will then be placed on the website under “resources.”

7.      YOUR NOTEBOOK MUST BE UPDATED ON A DAILY BASIS!!!  NO EXCEPTIONS!!!

8.      At the end of each month, I will review your notebook with you.

Purchases:

               All purchases are to be cleared through me or Jim; presently, only Jim has the authority to make purchases.

Oligonucleotides:

               Oligonucleotides are to be purchased through Operon using a standing PO number (see me for this number).  You can place the oligonucleotide order directly through their website at http://www.operon.com.  All oligonucleotides are to be designated by the initials of the user followed numerically by a number starting from 1 (for example, the first oligo I order is designated RM1, the second is RM2, the third is RM3, and so on).  Give a BRIEF one line description of the oligonucleotide (what is it used for?); this information is then submitted to the oligonucleotide database on the lab’s website.

Plasmids:

               At the end of each month, please submit to me a list of ALL plasmids you have constructed and verified (see below).  This list should include the plasmid name, map (made in MacPlasmap, or a similar mapping program for the PC), sequence file (in Strider or text format), and source (if you obtained it from someone else).  The plasmid database on the lab’s website can be searched for any of our cataloged plasmids.

               Verification of plasmids:  Plasmids must be verified in one of two ways:

1.      Sequence:  if a plasmid was constructed by insertion of an oligonucleotide or a PCR product, the entire inserted fragment must be sequenced to ensure that no sequence errors were introduced.

2.      Restriction mapping:  if a plasmid was constructed by subcloning a previously sequenced insert from another plasmid, then sequencing is not necessary.  Please be sure that you have mapped the plasmid by cutting with a variety of enzymes and have obtained the anticipated fragments.  A limited restriction mapping is ALWAYS necessary after maxi-prepping any plasmid.

Storage of plasmids:  Maxi-prepped plasmids must be stored in TE at either 4 C or –20 C.  If you choose to store cut plasmids (after restriction digestion), these must ALWAYS be stored at –20 C, and for no longer than one month (linearized DNA fragments are subject to exonuclease digestion, and their ligation potential decreases dramatically with storage time).

Bacterial and yeast strains:

               Presently this laboratory does not maintain a computerized database of bacterial and yeast strains.  Please maintain –80 C glycerol stocks (90% liquid growth medium, 10% sterile glycerol) of all strains you happen to acquire.  A computerized database is in the works.

Mammalian cell lines:

               This laboratory maintains a cataloged list of all mammalian cell lines.  Please see Jim for information on how to search for specific lines and how to catalog lines for storage.  A computerized database will soon come online.

               All cell lines are to be maintained by the person who grows them, including weekends and holidays.  If you can’t be around on specific days to pass your cell line, please ask someone else in the lab to do this for you (either me or Jim, for example) rather then letting the line die off.

               STERILITY AND CARE are of utmost importance when dealing with cell lines.  Please spray down the biosafety hood with 70% ethanol after all procedures, even the most minor.  Always use gloves when working with cell lines.

               The 37 C water bath chamber on the RIGHT is for warming mammalian cell culture media ONLY.  You can change the one on the left to whatever temp. you want.  The water for the chamber on the right must be STERILE water, supplemented with 0.5 ml of benzalkonium chloride per liter.  This chamber is to be cleaned at least every two weeks.

Enzymes:

               IT IS ABSOLUTELY IMPERATIVE that all enzymes be kept at –20 C at all times.  When you use an enzyme, place it in the –20 C freezer block, which is next to the enzyme blocks.  Take this block to your bench and aliquot the enzyme from it.  NEVER, EVER place any enzyme on ice.  The only exception to this is alkaline phosphatase, which is kept at 4 C and may be placed on ice.

Radioactivity:

               Radioactivity guidelines as set forth by EHS are strictly followed in this laboratory.  Please see my guidelines for use of radioactivity.  For all procedures involving radioactive source vials, please use the fume hood.

Always record the amount of isotope removed for an experiment on the appropriate inventory sheet AND survey the equipment and area where radioactivity has been used AND record the results of your survey in the radioactivity lab manual.

Lab Waste

Regular waste includes all non hazardous waste such as paper, plastic, cardboard etc., which can be discarded in the trash cans distributed throughout the lab.

Laboratory waste:  The brown cardboard receptacles labeled “laboratory waste” are used for solid lab waste which includes gloves, pipets, tips and tubes from cell culture experiments or other lab procedures like Maxipreps, restriction enzyme digests etc.  These containers should not contain any hazardous chemical or radioactive waste.  Anythong that is considered BSL-1 waste can be discarded in these containers.

Chemical waste:  White palstic buckets with “hazardous Waste” labels are used for solid waste like tips, pipets, gloves, and tubes that have come in contact with hazardous chemicals such as acrylamide, ethidium bromide, b-mercaptoethanol, phenol, chloroform, methanol, formaldehyde etc.  Liquid chemical waste containers are located in the Fume hood in room 2333.  Whenever possible, keep chemical waste separated by class or type (ie, do not mix oxidizers with aldehydes and /or benzene).  If you are not sure whether to mix liquid chemical waste, put them in separate containers.  New containers can be obtained by calling Environmental Health and Safety at 2-4911.

Radioactive waste:  Solid and liquid radioactive waste containers are located in room 2333.  Radioactive isotopes should be discarded in separate containers (ie, 3H in one container separate from 32P in another container).  In other words, do not mix isotopes.  Please advise Jim when waste containers are full and ready to be picked up or if new containers are needed for use with different isotopes. 

Regulated Medical Waste (CMC’s): cardboard boxes with red bag liners are used for viral vector waste, adenovirus cell cultures, and any human blood, tissue or cells.  Any item that is considered to require BSL-2 or BSL-3 safety precautions should be decontaminated and discarded in CMC’s.  When full, these containers should be sealed, labeled and taken to the Fontaine Loading Dock put in Room 1241.  The key for this room is in Jim’s to right desk drawer.

Each member of the lab should take responsibility for compacting and/or sealing lab waste containers when they appear to be overflowing and set up new replacement containers.  Jim will be responsible for calling in all chemical and radioactive waste material for pick up from the lab.