K-12 Programs

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Restriction Enzyme Digest Field Trip
Set-up

I. Reagents needed:

Each student prepares one reaction tube, set up 4 students/4 different digests per bench. Each group prepares a negative control.

1. Nuclease-free water: 1 bottle per bench
2. Clear 1.5 mL Eppendorf tube: 5 tubes per bench
3. 6x loading dye tube: 1 tube per bench
4. Pink 0.5 mL Eppendorf tube containing 7 µL of unmethylated lambda DNA (60ng/µl).  Aliquoted DNA may be stored in the refrigerator: 2 tubes per bench
5. Clear 0.5 mL Eppendorf tube containing 10 µL of EcoRI/Hind III Marker (100ng/µL) **
6. Green 0.5 mL Eppendorf tube containing 5 µL of  Buffer J
7. Yellow 0.5 mL Eppendorf tube containing 5 µL of Buffer B
8. Blue 0.5 mL Eppendorf tube containing 5 µL of Buffer H
9. Orange 0.5 mL Eppendorf tube containing 5 µL of Buffer E
10. Aliquot the four different enzymes (SmaI, Eco RI, Hind III, EcoRI/HindIII): These enzymes are stored in the freezer until they are ready to use.  Aliquot as close to time for use as possible as the small volumes evaporate readily.  

1. Prepare 0.8% agarose gel:  Weight 0.8 g of agarose and add it to a 250ml flask containing 100 ml of 0.5x TBE. (Agarose may be allowed to hydrate in the TBE buffer overnight.). Prepare 35 ml of 0.8% agarose for each gel (4 students per gel).  Microwave the agarose or heat it in a water bath at 55-60^oC so it will be in solution.  Add 5µl ethidium bromide per 100mls of agarose when the agarose has cooled to about 55^oC.  Gels may be poured in advance and stored in a small amount of buffer in the refrigerator.
2. 0.5 x TBE buffer (300-350 mls per electrophoresis chamber); 1 electrophoresis chamber per bench. ***
3. EtBr (10mg/ml stock): 1 bottle per lab, keep this bottle in refrigerator until it is ready to use (see step 1).

II. Equipment set up:

1. P20 pipetteman with small pipette tips: 2 per bench
2. Mini table top centrifuges
3. Ice bucket: 1 per bench
4. Heat block set at 37°C 
5. 1 Water bath per lab set up at 55°C (for melted agarose.  Not necessary if gels are poured in advance).
6. Gel electrophoresis set up:
   1. gel box (1 per group)
   2. gel tray with one 6-well comb (1 per group)
   3. power supply (1 per two gels)

**Prepare EcoRI/Hind III Marker (100ng/mL) from the stock EcoRI/Hind III Marker (500ng/mL) by using 100 µL of stock (500ng/mL) then dilute with 315 µL ddH₂0 and 85 µL 6x loading dye (final volume 500µL).  Store in freezer. 

***Prepare 0.5x TBE buffer from 10x TBE by using 200 mL of stock (10x) then dilute with 3,800 mL ddH₂0 (final volume 4 L).

Note:  To increase longevity of equipment, rinse gel boxes, trays and combs with distilled water if possible.  

• Background
• Protocol
• Biotechnology Field Trips Home Page

Contact: Barbara Bielec, 608-273-9737