Restriction Enzyme Digest Field Trip

Setup

Note: Each student prepares one reaction tube, set up 4 students/4 different digests + 1 negative control per bench.

I. Reagents needed:

1. Nuclease-free water: 1 bottle per bench
2. Clear 1.5 mL Eppendorf (Ep) tube: 5 tubes per bench (4 reactions, 1 control)
3. 15µl FOTO/Vision^TM per bench
4. One ice bucket per bench with the following reagents (one tube per bucket):




Tube (Color and Size)	Tube Label	Contents
Pink 0.5ml Ep	λ	30µl methylated λ DNA* (60ng/µl)
Purple 0.5ml Ep	BB	8µl Restriction Buffer B
Clear 0.5ml Ep	marker	6µl Lambda EcoRI/HindIII Marker**
Green 0.5ml Ep	PstI	2.5µl PstI
Yellow 0.5ml Ep	E/H	1.5µl EcoRI /1.5µl HindIII
Blue 0.5ml Ep	EcoRI	2.5µl EcoRI
Orange 0.5ml Ep	HindIII	2.5µl HindIII

If pouring agarose gels for students in advance of the laboratory:
 

5. Prepare 0.8% agarose gel:  Weigh 0.8g of agarose and add it to a 250ml flask containing 100 ml of 1x sodium borate buffer. (Agarose may be allowed to hydrate in the buffer overnight.). Prepare 35ml of 0.8% agarose for each gel (4 students per gel). Microwave the agarose or heat it to a boil so that the agarose goes into solution. Gels may be poured when the agarose has cooled to about 55^oC. Gels may be cast the night before class and stored in a small amount of buffer in the refrigerator.
6. Each electrophoresis chamber will require 350 to 350ml running buffer.

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. Gel electrophoresis set up:
   • gel box (1 per group)
   • gel tray with one 6-well comb (1 per group)
   • power supply (1 per two gels)

*Stock, Methylated λ:
Lambda DNA is a product that can be ordered from Promega at 420ng/µl.
Dilute, Methylated λ DNA is created by diluting the concentrated stock sevenfold (1 part Stock DNA: 6 parts nuclease-free water) to arrive at a 60ng/µl solution that is dispensed for student use.

**Lambda EcoRI/HindIII Marker:
To prepare 60µl stock tube of Promega Lambda EcoRI/Hind III Marker:
Combine 10µl Promega Lambda DNA/EcoRI + HindIII marker (500ng/µl) with 40µl

Buffer B diluted to 1x.
Dispense 5µL of the diluted marker into 0.5ml tubes and add 1µl FOTO/Vision^TM dye. The entire 6µl should be loaded onto the gel as a marker.

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