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Chapter 20

microcentrifuge (optional)

37 °C water bath

thermometer

1 g agarose

paper boat

electronic balance

500 mL Erlenmeyer flask

250 mL graduated cylinder

microwave or hot plate

flask tongs or oven mitts

gel casting tray and gel electrophoresis box

1L 1TBE buffer

5 L loading dye

power supply (45 V)

plastic wrap

25–30 mL 0.025 % methylene blue, or enough to cover the

gel in the staining tray

light box or overhead projector

acetate sheet

Wear safety goggles at all times.

Wear gloves when performing the experiment.

Wipe down all surfaces with 70 % ethanol, or 10 %

bleach, before and after the laboratory exercise.

Do not use ethanol near a heat source.

Wash your hands thoroughly at the end of the

laboratory.

Procedure

Day 1: Restriction Enzyme Digestion

1. Put on your safety goggles and gloves, and wipe
   down your bench with a 70 % ethanol solution
   (or 10 % bleach).

INVESTIGATION 20.2continued

Table 1 Reagents to Add to Tubes

Tube DNA 10  Water BamHI EcoRI HindIII

(L) buffer (L) (L) (L) (L)

(L)

BamHI 41 4 1

EcoRI 41 4 1 

HindIII 41 4 1

control 41 5 

Ethanol is highly flammable. Make sure that any

flame on your desk or near it is turned off before use.

2. Label four 1.5 mL Eppendorf tubes “BamHI,”
   “EcoRI,” “HindIII,” and “control.” Place the tubes in a
   polystyrene cup containing crushed ice.Table 1
   outlines the amount of reagents to add to each tube.
   To keep track of each tube’s contents, copy the table
   into your notebook and check off each reagent as you
   add it to the tube.


3. Read down each column, adding the same reagent to
   all appropriate tubes. Use a fresh tip on the
   micropipette for each reagent. Add the 4 L of DNA
   to each tube first, followed by the 10reaction
   buffer, and then the water.Make sure you add the
   enzyme last. Dispense all the contents close to the
   bottom of the Eppendorf tubes. Ensure that the
   pipette tip is touching the side of the tubes when
   dispensing the contents.Keep everything on ice at all
   times.


4. Close the Eppendorf tube tops. Place the tubes in the
   microcentrifuge, close it, and spin at maximum speed
   for approximately 3 s. If you do not have access to a
   microcentrifuge, then just tap the tubes on a soft pad
   or thick paper towel on the bench, pooling the
   contents to the bottom.

NEL Molecular Genetics 697

0

5 505

EcoRI EcoRI EcoRI EcoRI EcoRI

31 747 34 499 39 168 41 732

48 500

23 132

21 226

22 346

HindIII HindIII HindIII

BamHI BamHI BamHI BamHI BamHI

37 495

36 895

44 972

44 141

27 479

25 157

26 104

27 972 37 584

Figure 1
Restriction enzyme map of bacteriophage lambda DNA