John Ohlrogge , Mike Pollard Lab Manual

Full pdf version (106 pages)

TABLE OF CONTENTS (Rev. May 2003)

Please use I.E. version 6 or better when possible.

I. Information for ‘New Arrivals to Lab’................................................................................... I-1

A. SOCIAL SECURITY NUMBER (SSN) ..................................................................... I-1

B. MSU IDENTIFICATION CARD................................................................................ I-1

C. PARKING PERMIT .................................................................................................. I-1

D. E-MAIL ACCOUNT................................................................................................... I-2

E. GREENHOUSE ACCESS........................................................................................... I-2

F. ORCBS (Office of Radiation, Chemical and Biological Safety) training............................ I-2

H. LIBRARY INFORMATION ...................................................................................... I-2

Circulation.............................................................................................................. I-3

Database searching ............................................................................................... I-3

Where to find journals............................................................................................. I-3

What to do if MSU doesn’t own what you need ..................................................... I-4

List of commonly used journals and where to find them ........................................... I-5

I. HOW TO USE THE OHLROGGE LITERATURE DATABASE AND REPRINT COLLECTION I-8

J. NON-DEPARTMENTAL FACILITES IN THE PLANT BIOLOGY BUILDING
.............................................................................................................................. I-8

Dry ice & liquid nitrogen......................................................................................... I-8

Phosphoimager....................................................................................................... I-8

X-ray film development machine.............................................................................. I-8

Autoclaves.............................................................................................................. I-9

Computer room...................................................................................................... I-9

Growth chambers................................................................................................... I-9

Overhead projectors/Slide projectors...................................................................... I-9

Photocopier & Duplicator/Collator.......................................................................... I-9

L. HOW TO ORDER THE SUPPLIES YOU NEED......................................................... I-9

Summary: How to order....................................................................................... I-10

M. MISCELLANEOUS IMPORTANT INFORMATION............................................. I-11

Dialing on and off campus phone numbers............................................................. I-11

Common abbreviations......................................................................................... I-11

What is the MSU PRL-DOE anyway?.................................................................. I-11

II. Ohlrogge Lab Information.................................................................................................... II-1

A. CONTACTING THE OHLROGGE LABORATORY................................................ II-1

Ohlrogge Lab address........................................................................................... II-1

B. SUPPLIES INVENTORY.......................................................................................... II-1

C. FREEZER SPACE INVENTORY.............................................................................. II-1

D. USING THE NETWORKED COMPUTERS.............................................................. II-2

Computer networking and personal files................................................................. II-2

Instructions for accessing your personal file space on drive 2 of Double Drive computer II-2

Trouble-shooting................................................................................................... II-3

E. VERY IMPORTANT RULES FOR PRESENTATIONS AT LAB MEETINGS OR OTHER SCIENTIFIC MEETINGS.......................................................................................................... II-3

F. RELEVANT MISCELLANEOUS INFORMATION.................................................. II-4

How to use pipettors............................................................................................. II-4

Safety Glasses....................................................................................................... II-4

Some useful units and conversions.......................................................................... II-4

Arabidopsis Genome............................................................................................. II-5

G. RADIOACTIVE WASTE DISPOSAL/STORAGE/PICKUP, SURVEYS, PURCHASING, RECEIVING............................................................................................................................. II-5

Waste disposal/storage/pickup............................................................................... II-5

Request a pick up.................................................................................................. II-7

Surveys................................................................................................................. II-7

Purchase & receipt of radioisotopes....................................................................... II-7

H. ANTIBODIES AVAILABLE IN THE OHLROGGE LAB......................................... II-8

I. PLANT GROWTH FACILITIES & CARING FOR YOUR PLANTS........................ II-9

Controlled environment options.............................................................................. II-9

Non-controlled environment options....................................................................... II-9

What care of your plants entails.............................................................................. II-9

Disposing of your plants....................................................................................... II-10

Good greenhouse hygiene.................................................................................... II-10

J. LAB PUBLICATIONS: 1995-2000 .......................................................................... II-11

III. Equipment Operation Instructions.................................................................................... III-1

A. Instructions for DIGITAL CAMERA.......................................................................... III-1

Capturing Images................................................................................................. III-1

Replacing thermal paper rolls................................................................................ III-1

B. Instructions for LIQUID SCINTILLATION COUNTER............................................. II-3

Beckman LS 6500 Liquid scintillation counter (room 362)...................................... II-3

Sample preparation.................................................................................... II-3

Operating controls..................................................................................... II-3

Edit user program...................................................................................... II-4

Starting the counting................................................................................... II-4

Stopping the counter.................................................................................. II-4

Quick Guide to LS 600 Data Capture/Network software........................... II-4

Counting rules................................................................................ II-5

C. Instructions for PACKARD INSTANT IMAGER...................................................... III-4

New Users........................................................................................................... III-4

Regular use.......................................................................................................... III-4

Notes.. III-5

D. Instructions for pH METER....................................................................................... III-6

Preliminary operations.......................................................................................... III-6

Single buffer standardization.................................................................................. III-6

Two buffer standardization.................................................................................... III-6

E. Instructions for the SPECTROPHOTOMETER ......................................................... III-7

General Information.............................................................................................. III-7

Lamp warm-up.................................................................................................... III-7

Changing the cuvette holder.................................................................................. III-7

Re-alignment........................................................................................................ III-7

Measurement programs........................................................................................ III-7

Printing III-8

An example of how to use the spec and software: Fixed Wavelength measurements III-8

F. Instructions for SPEED-VAC...................................................................................... III-9

Start up................................................................................................................ III-9

Shut down............................................................................................................ III-9

IV. Lab Duties Described........................................................................................................ IV-1

A. MAINTAINING LAB SUPPLIES............................................................................ IV-1

B. INSTANT IMAGER MONITOR DUTIES............................................................... IV-2

Counting gas........................................................................................................ IV-2

Calibrations.......................................................................................................... IV-3

Users and user files.............................................................................................. IV-3

Packard Instant Imager information....................................................................... IV-4

C. GREENHOUSE DUTIES.......................................................................................... IV-5

D. PLANT COORDINATOR DUTIES......................................................................... IV-6

E. RADIOACTIVE AND HAZARDOUS WASTE MONITORS.................................. IV-7

V. Safety and Emergencies....................................................................................................... V-1

A. PERSONAL INJURIES/WORKERS’ COMPENSATION....................................... V-1

Emergencies....................................................................................................... V-1

Non-emergencies.................................................................................................. V-1

B. CHEMICAL AND RADIATION SAFETY................................................................ V-1

CHEMICAL SAFETY......................................................................................... V-1

STANDARD OPERATING PROCEDURES........................................... V-2

GENERAL SAFETY PRINCIPLES......................................................... V-2

HEALTH AND HYGIENE...................................................................... V-3

FOOD AND DRINK IN THE LABORATORY...................................... V-3

HOUSEKEEPING................................................................................... V-3

CHEMICAL HANDLING AND STORAGE........................................... V-4

TRANSFERRING OF CHEMICALS...................................................... V-4

COMPRESSED GASSES........................................................................ V-5

UNATTENDED OPERATIONS............................................................. V-5

WORKING ALONE............................................................................... V-6

STORAGE AND DISPOSAL OF HAZARDOUS WASTE..................... V-6

RADIATION SAFETY........................................................................................ V-6

General Rules for Radiation Safety............................................................. V-6

C. OOPS! I SPILLED SOME TOXIC/RADIOACTIVE CHEMICAL!
............................................................................................................................. V-7

EMERGENCY AND SPILL RESPONSE........................................................... V-7

BASIC STEPS FOR EMERGENCY AND SPILL RESPONSE.............. V-7

Emergency Situation - Fire............................................................. V-8

Emergency Situation - Spill............................................................ V-9

Mercury Spills............................................................................. V-10

Spill Kits..................................................................................... V-10

Non-Emergency Situation - Spill.................................................. V-10

Power Outages........................................................................... V-11

HANDLING RADIOACTIVE INCIDENTS/EMERGENCIES......................... V-12

MINOR incident..................................................................................... V-12

MAJOR incident..................................................................................... V-13

EMERGENCY....................................................................................... V-13

D. LOCATIONS OF SAFETY AND EMERGENCY EQUIPMENT IN THE OHLROGGE LAB V-14

VI. Protocols............................................................................................................................. VI-1

A. TIPS FOR LIPID RESEARCH................................................................................. VI-1

Suggested readings............................................................................................... VI-1

General Tips: Logistical....................................................................................... VI-1

General Tips: Chemical......................................................................................... VI-2

Use of the Fume Hood......................................................................................... VI-3

B. LIPID EXTRACTIONS............................................................................................ VI-5

General Principles................................................................................................ VI-5

Notes.. VI-5

Literature............................................................................................................. VI-5

Hexane-Isopropanol Method............................................................................... VI-6

Chloroform-Methanol Methods............................................................................ VI-6

Use of Evaporator/nitrogen Gas Tanks................................................................. VI-7

C. PREPARATION OF FATTY ACID METHYL ESTERS (FAMEs).......................... VI-8

General notes....................................................................................................... VI-8

Acid catalyzed esterification and transesterification................................................ VI-8

Boron Trichloride Methanol...................................................................... VI-8

Esterification/transesterification with Methanolic:HCl................................. VI-9

Base-Catalyzed Transesterification..................................................................... VI-10

D. THIN LAYER CHROMATOGRAPHY (TLC) OF LIPIDS.................................... VI-10

Introduction ...................................................................................................... VI-10

General notes..................................................................................................... VI-11

Types of TLC plates.......................................................................................... VI-11

Solvents and solvent systems.............................................................................. VI-13

Literature........................................................................................................... VI-13

Neutral lipids...................................................................................................... VI-13

Polar lipids......................................................................................................... VI-13

Fatty acid methyl esters (FAME) by reversed-phase TLC................................... VI-14

Fatty acid methyl esters (FAME) by silver nitrate (argentation) TLC.................... VI-14

Monoene separation. ............................................................................. VI-14

Detection of lipids on TLC plates........................................................................ VI-14

Direct Visualization Under UV Light....................................................... VI-14

Staining with iodine vapor....................................................................... VI-15

Phosphomolybdic acid........................................................................... VI-15

Dichlorofluorescein................................................................................. VI-15

α-Naphthol for glycolipids...................................................................... VI-15

Radioactivity.......................................................................................... VI-15

E. SIMPLIFIED ARABIDOPSIS TRANSFORMATION PROTOCOL..................... VI-15

F. TRANSFORMATION OF ARABIDOPSIS BY VACUUM FILTRATION........... VI-17

References......................................................................................................... VI-19

Solutions............................................................................................................ VI-19

Commonly asked questions about this vacuum infiltration method........................ VI-20

G. DEVELOPMENT OF WESTERN (PROTEIN) BLOTS......................................... VI-21

Blot preparation/fixing........................................................................................ VI-21

Blot development............................................................................................... VI-21

Buffer and stock solutions................................................................................... VI-22

H. PROTEIN ASSAY.................................................................................................. VI-23

General Principles.............................................................................................. VI-23

Method.............................................................................................................. VI-23

I. ACP ASSAY USING ACYL-ACP SYNTHETASE REACTION............................ VI-24

Preparation of fatty acids for acyl-ACP synthetase reaction................................. VI-25

Acyl-ACP Synthetase Stock Solutions............................................................... VI-25

J. PROTOCOL FOR ISOLATING CHLOROPLASTS FROM SPINACH, PEA AND AMARANTHUS VI-26

Growing appropriate plants................................................................................ VI-26

Isolation Buffers.................................................................................................. Vi-27

Isolation Procedures........................................................................................... Vi-29

Incubation Buffers............................................................................................... Vi-30

Assays Vi-30

Additional Information........................................................................................ VI-33

K. USE OF PAT GENE AS SELECTABLE MARKER FOR PLANT TRANSFORMATIONS
.......................................................................................................................... VI-33