Laboratory Equipment | 8 | v2 | DNA Fingerprinting in Plants

ABSTRACT

Required for tissue culture and/or molecular cloning experiments

HYB

Required for experiments that involve blot hybridization

RAD

Required for experiments that involve the use of radioisotopes

3.1 INCUBATORS

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Water baths (e.g., for growing plant cells, labeling probes, blot hybridization, restriction, ligation, incubation of extracts in some DNA isolation methods)

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Thermal cycler (for automated PCR reactions; heating of probes and samples)

Thermostat-controlled metal heating blocks, which hold test tubes and/or microcentrifuge tubes (for small-scale DNA extraction, incubation, DNA restriction)

CLON, HYB

Dry incubator or rotary shaker (e.g., for growing bacterial cells, blot hybridization, washing Southern blots, staining agarose gels)

HYB

Hybridization oven for rotating tubes (for Southern blot and gel hybridizations; sealed bags in a thermocontrolled rotary shaker or water bath can also be used)

3.2 PLANT AND PLANT CELL GROWTH EQUIPMENT

CLON

Sterile bench, laminar flow hood, and/or biological safety cabinet (e.g., for micropropagation)

CLON

Constant-temperature room available: a rotary shaker with clamps for 100-, 250-, 500-ml and 1-to 2-liter Erlenmeyer flasks

CLON

Constant-temperature room not available: a closed-environment rotary shaker equipped as above

3.3 STERILIZATION

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Autoclave, leak-proof autoclaving bags, sterilization tape (to label sterilized solutions, glassware, or other items)

xxx

Filters and filtration units (0.22-µm pore size; to sterilize solutions that cannot be autoclaved, e.g., vitamins, glucose)

Oven (e.g., to sterilize glassware)

Ultraviolet (UV) light source

CLON

Bunsen burner or equivalent (to sterilize inoculation loops)

3.4 WATER PURIFICATION

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Deionization or water purification cartridges and/or glass distillation apparatus

Storage containers

3.5 CENTRIFUGES

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One or more microfuges (optionally refrigerated) that hold standard 1.5-ml and/or 0.5-ml microfuge tubes and/or microtiter plates (for small-scale DNA extraction, PCR, and many other applications). The centrifuges should be equipped with both fixed angle and swinging-bucket rotors

Low-speed centrifuge (10,000 to 20,000 rpm) floor model, or tabletop-refrigerated centrifuge for larger tubes (for large-scale DNA extraction)

Vacuum concentrator (e.g., SpeedVac

, for concentrating DNA solutions)

x, HYB

Ultracentrifuge (20,000 to 80,000 rpm) with fixed-angle, vertical, and/or swinging-bucket rotors (for large-scale isolation of highly purified DNA via CsCl gradients)

3.6 REFRIGERATION AND MATERIAL STORAGE

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Refrigerator 4˚C (to store, e.g., solutions, DNA, PCR products)

xxx

Freezer –20˚C (to store, e.g., solutions, aliquots of PCR reagents, deoxynucleotide triphosphates [dNTPs], DNA,

Taq

DNA polymerase, restriction enzymes, and other DNA-modifying enzymes)

Freezer –70 to –80˚C (for long-term storage of frozen tissue samples, reagents, DNA, bacterial cells, X-ray cassettes for autoradiography)

Isothermic storage containers for liquid nitrogen (needed for many DNA isolation procedures) and dry ice

A cold room is very useful for performing reactions and centrifugations at 4˚C

Ice machine

Lyophilizer (to freeze-dry plant material, e.g., for long-term storage)

3.7 SAFETY

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Laboratory coats and gloves

xxx

Proper UV shielding and UV-protective glasses

xxx

Fume hood

xxx

Proper storage and containment systems for collection of hazardous waste, such as ethidium bromide, organic chemicals, and radioisotopes

RAD

Plexiglas radiation shield and containers (essential when working with strong

-emitters such as

RAD

Contamination monitor (Geiger counter)

3.8 PIPETS

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Adjustable micropipets and disposable pipet tips (e.g., 0.5 to 10, 10 to 100, 100 to 1000; 1000 to 5000

l). If possible, one set of pipets should be designated for each full-time researcher and one set for pre-PCR work exclusively. Pipets or pipet tips can be plugged with cotton on one side to prevent cross-contamination by aerosols. Plugged tips are also commercially available

xxx

Glass or disposable plastic pipets (0.2, 1.0, 5.0, 10.0, and 25.0 ml)

Pasteur pipets

One-channel or multichannel micropipets (e.g., Hamilton pipets) for loading ultrathin polyacrylamide gels

Mechanical (Peleus

balls) and/or electrical pipetting devices for glass pipets

3.9 SUPPLIES (GLASSWARE AND PLASTICWARE)

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Common laboratory glassware and plasticware including beakers, graduated measuring cylinders, Erlenmeyer flasks, reagent bottles of various sizes

xxx

PCR tubes and caps, usually in strips of eight or plates of 96

xxx

Microtiter plates with and without caps (e.g., for PCR, preparing samples for gel loading, DNA cloning)

xxx

Microfuge tubes and caps (0.2, 0.5, 1.5, and 2.0 ml)

Glass or polypropylene centrifuge tubes and adapters (e.g. Corex

tubes; 15 and 30 ml)

Polypropylene snap-cap and/or sterile screw-cap tubes (15 and 50 ml; for organic extractions)

Polyallomer or ultraclear ultracentrifuge tubes (for ultracentrifugation only)

CLON

Culture tubes and caps

CLON

Petri dishes

3.10 DNA DETECTION AND QUANTITATION

Spectrophotometer (UV and visible light)

Fluorometer

Portable UV source (254 nm)

3.11 ELECTROPHORESIS EQUIPMENT

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Power supply: medium voltage (up to 500 V) for agarose gels

xxx

One or more submarine horizontal gel apparatus (at least 15

20 cm; preferably 25

30 cm) for agarose gels

Submarine minigel apparatus for agarose gels

UV-transparent trays (for photographing stained gels)

Automated DNA sequencer including the appropriate software for fragment analyses (for DNA sequencing and fluorescent fragment analysis, including amplified fragment length polymorphisms [AFLPs] and microsatellites). Note: If an automated sequencer is available, there is no need to buy the standard polyacrylamide gel

electrophoresis equipment listed below. Such an instrument also circumvents the use of isotopes for fragment analyses and DNA sequencing

Vertical gel apparatus for polyacrylamide gels (e.g., for AFLP and microsatellite analysis)

High-voltage power supply (up to 3000 V) for polyacrylamide gels

Combs, spacers, glass plates, clamps, and/or gel sealing tape for polyacrylamide gels

RAD

Gel dryer and vacuum pump (for drying radioactive polyacrylamide gels)

HYB

Vacuum transfer unit or electrotransfer unit (optional; can be used as a quicker alternative to the classical Southern blot technique)

3.12 DOCUMENTATION OF RESULTS

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Gel documentation system consisting of a UV transilluminator (302-or 366-nm wavelength; to visualize DNA fragments), a digital camera attached to a computer and a (thermo)printer, UV-protective shielding, and appropriate filters for UV photography (red filter for ethidium bromide-stained gels). The individual components can also be purchased separately. Alternatively, the older method for gel documentation by Polaroid photography can also be used.