Lab Guidelines

           

 

Please feel free to come to me AT ANY TIME with research related questions or help.  Please freely share equipment and chemicals with other trained members of this laboratory and other laboratory.  However, if equipments / reagents need to be borrowed by other laboratories, please note down the name and phone number of specific lab member and record the details in the specified laboratory log-book.  Plasmids, radioactivity, and human cell lines DO NOT leave this laboratory without my prior e-mail consent.

Laboratory Benchwork:

Wear appropriate personal protection equipment (PPE) when working in the tissue culture rooms or at your bench.  Labcoats and other PPE should be removed when leaving the laboratory and should not be worn in the corridors or break rooms.  Make sure to remove gloves while using your personal computer or the telephones in the labs. 

All reagents that are being used or stored in the refrigerator / freezer should be properly labeled / coded and should carry the name and date of the person who prepared this.  Any unlabelled tubes, vials or plates can be found in the trash bin.

Laboratory Notebook:

        I will provide you with a specific notebook.  The pages of this notebook are pre-numbered 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 laboratory.  The first page should be left blank and should carry an outline of the contents of the book.  Each experiment should start on a fresh page and should specify the “Purpose” and “Approach” (which may be described as text or cartoons).  Pictures of gels, animals, cells, etc. may be taken in duplicate, only if an electronic recording option is not available and if you desire a copy for your own duplicate pages.  Otherwise all pictures must remain in the original (white) pages of the notebook. 

        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 “Standard Operating Protocols”.  Your notebook must be updated on a daily basis!!!  No exceptions!!!  I will go through your lab notebook with you at the end of each month.

Purchases:

                All purchases are to be cleared through me.

Cell lines:

This laboratory maintains primary cultures and cell lines in separate incubators that are designated for human primary cultures or cell lines.  Do not place cell lines in incubators designated for primary cultures.  Rodent, monkey and chick cultures are maintained in the rodent animal cell culture room.  Cell lines available from the NCCS repository should be requested with sufficient request time.  All requests should be submitted generally 2 weeks prior to the date needed using the cell line request form signed by me.  Please see me for information on how to catalog cells and cell lines maintained in this laboratory for storage.  An Excel spreadsheet is available on the central lab PC and all cryostorage vials should be logged in this file.

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 maintain your cell line, please ask someone else in the lab to do this for you rather than 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 every procedure, even the most minor one.  Always use gloves when working in the biosafety hood.  Hood #1 in human cell culture room is meant for handling of primary human cells received by the lab.  Please DO NOT use this hood for any other cell types.  It is also a good idea to use double gloves especially when handling primary human cell samples.  Remove and dispose off all biological waste into designated double bagged biological waste containers and store at -20°C till incineration.  Use the freezer compartment on top of the refrigerators for storage of biological waste only.  These frost-free freezer compartments should NOT be used for storage of lab reagents / enzymes.     

The 37°C water bath chamber near the tissue culture room is for warming mammalian cell culture media ONLY.  You can change the one on the left to whatever temperature you want.  The water in all waterbaths must be distilled water, supplemented with 0.5 ml of benzalkonium chloride per liter.  It is everyone’s responsibility to take care that the water levels are maintained and the waterbaths are cleaned on rotation every month.

All new laboratory personnel are recommended to watch this video (Kindly provided and generated by ESCO as a training tool) for orientation to cell culture technique in the lab.

 

Enzymes:

IT IS ABSOLUTELY IMPERATIVE that all enzymes should 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 wet ice.  The only exception to this is alkaline phosphatase, which is kept at 4°C and may be placed on ice.

 

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). 

                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).

 

Radioactivity:

Radioactivity guidelines as set forth by NCCS Radiation Safety are strictly followed in this laboratory.  Please see me for detailed 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.  Use the pipettes and tips that are marked “Radioactive” and kept in the Radioactive fume hood.  These pipettes and tip boxes should not be taken out of the fume hood for routine bench work.