Restriction Enzyme Digest Field Trip--Protocol

K-12 Programs

Restriction Enzyme Digest Field Trip
Protocol

I. Reaction Set-up

Prepare these reactions in 1.5 ml Eppendorf tubes and incubate at the enzyme's preferred temperature for 45 minutes.

Each group should label 5 tubes with initials and the enzyme used, or "neg" for negative control.

ADD IN ORDER:

5 µl Lambda DNA (60 ng/µl) 1 µl 10x reaction buffer (Buffer____) 3 µl ddH₂O 1 µl Enzyme (Enzyme used________) 10 µl total volume	Enzyme	Buffer	Temp °C	Recognition Site
	EcoRI	H	37	G¯ AATTC
	EcoRI HindIII	B	37	G ¯ AATTC A ¯ AGCTT
	SmaI	J	25	CCC¯ GGG
	HindIII	E	37	A¯ AGCTT
Negative Control:	Water	Any	25

Incubate: Enzyme _________is incubated at__________°C.

II. Set up gel tray and pour gel

To prepare a 0.8% agarose gel, add 0.8g of agarose to 100 mls of 0.5x TBE. The agarose is allowed to hydrate in the buffer before the mixture is microwaved to dissolve the agarose. The agarose is then cooled to 55° C before pouring the gel.

NOTE: EtBr is a mutagen; it enters the cell, intercalates into the DNA and mutates it. Always wear gloves, lab coat and safety glasses when using EtBr.

Prepare the gel tray by bringing up the dams at the ends of the tray and tightening the screws firmly, but not too tightly. The screws tend to snap.
Place the 6-well comb into the slots at the top of the gel.
Add 5 µl EtBr to 100 ml of 55°C agarose.
Pour the agarose into the middle of the tray until it is about half way up the teeth of the comb and has filled the tray to the corners. Do not disturb the tray while the agarose is solidifying (about 20 mins.).

III. Set up gel box with buffer and gel

After the gel has set, loosen the screws and lower the dams. Hold the tray on the high side, with the wells closest to the black (negative) electrode, and slip it into the electrophoresis chamber on top of the platform. The dams will hang down over the ends of the platform.
Rock the comb very gently back and forth in the gel. Gently remove the comb. Remember the gel contains EtBr.
Add enough 0.5x TBE to the electrophoresis chamber to just cover the gel, about 325-350 mls.

IV. Load gel and run

Controls for the gel:

1. After incubation, add 2 µl of the 6x Loading dye to each reaction and the negative control.

2. Fill in the table listing the order of the samples loaded on the gel. (Lane one is the well closest to you as you load.)

Lane Sample

1 10 µl Negative Control

2_______________________________

3_______________________________

4_______________________________

5 10µl EcoRI/HindIII – Lambda DNA Marker

6_______________________________

3. Load the controls (10µl Negative Control and 10µl EcoRI/Hind III-lambda marker). Load 10µl of each digest into the appropriate well.

4. Place the lid on the gel box; connect the electrodes to the power supply. Make sure that the black wire goes into the black plug and the red into red.

5. Turn on the power supply and set it at 130 V. Bubbles at the electrodes indicate that there is a current. The gel will run for approximately 45 minutes.

V. View gel

After the gel has run, remove the gel from the gel box. Drain as much buffer as possible off of the gel. Remember the gel contains EtBr. If your gloves are wet from handling the gel, change them before handling anything else in the lab. Place it on the UV light box to visualize the DNA and to photograph the gel.

VI. Analysis

My restriction enzyme was __________. It has ________ restriction sites in lambda DNA

resulting in ______ DNA fragments after digestion. The lengths of the fragments are (in

base pairs): ____________________________________________________________

The negative control for this experiment is__________________ and demonstrates

_____________________ conditions. The molecular weight marker is used to

_____________________________________________________________________

FOR MORE INFORMATION

Contact: Barbara Bielec, 608-273-9737

K-12 Programs