Pre-Laboratory Preparation

Kit Materials:

See complete materials list for this experiment at the end of this manual.

This kit provides enough material for a class of 30 students (6 groups; 5 students/group).

DNA Samples:

lambda DNA / EcoR I digest (2 x 10 ug)

lambda DNA / Hind III digest (2 x 10 ug)

lambda DNA Hind III/ EcoR I double digest (2 x 10 ug)

Note: Each 10 ug sample contains enough DNA to run 8 lanes. Therefore, each sample type contains enough DNA to run a total of 16 gel lanes.

1	Bottle, 0.8% pre-cooked agarose (200 mL)
1	Bottle, TBE running buffer (10X) (250 mL)
2	Bottles, Ward's DNA Stain (500 mL)
18	Microfuge "reaction" tubes
2	Gel staining trays
1	Spatula ("gel handler")
30	Sheets, graph paper
1	"Student Study Sheet" copymaster
1	"Student Analysis Sheet" copymaster
1	Teacher's Manual

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Pre-Laboratory Preparation Procedure

(1)	Prepare TBE Running Buffer (1X) according to the following directions: to 20 mL TBE running buffer concentrate (10X) add 180 mL distilled water. This will make a working buffer solution of 1X. This 200 mL quantity is enough for 1 gel.
(2)	Cast 0.8% agarose gels. Refer to Section 2.3 in "Ward's Agarose Electrophoresis Manual" or product literature supplied with pre-cooked agarose. Each student team will run 3 sample lanes (lambda DNA EcoR I; lambda DNA Hind III, lambda DNA EcoR I / Hind III double digest). Suggested run formats below:

Student Teams
(5 students / team)	No. Gels (6-well)	No. Gels (12-well)
1	1	1
2	1	1
3	2	1
4	2	1
5	3	2
6	3	2

Additional Prep Notes:

(3)	In this experiment 3 DNA samples will be electrophoresed. It is suggested that each student team receive their own set of labeled DNA samples prepared in advance by you. Prelabel student microfuge "reaction" tubes (set of 3 tubes per student group) as follows: Tube #1 "lambda DNA EcoR I digest" Tube #2 "lambda DNA Hind III digest" Tube #3 "lambda DNA EcoR I / Hind III double digest"
(4)	Using a micropipet (see "micropipeting tips"), transfer EXACTLY 10 uL of each of the above sample types into corresponding reaction tubes. (Use of a microfuge tube rack is recommended.) Refreeze any unused DNA sample.
(5)	Heat Tube # 2 at 65deg. C for 5 minutes. Heating helps band imaging. Note: Use the gray sample container as a "micro water bath." Place tube #2 into the container and pour water (90deg. C) up to the dividers. Close the cover. Allow to stand 5 minutes. Average incubation temperature will be 65deg.C).
(6)	Use the enclosed Ward's copymasters ("Student Study" and "Student Analysis") to make worksheet copies. Permission is granted for unlimited copies made for use within any single school building.
*	You may wish to designate one area of the laboratory as an "electrophoresis area" where cells and power supplies are connected. Assure that any wall outlet is properly wired; i.e., that correct polarity exists (use a circuit tester). Assure that electrical power cords and patch cords are clear from moisture or wetness.
*	Assure that all students are thoroughly drilled on the correct procedure regarding making electrical connections and that they are directly supervised by you.
*	Assure that all students wear the following personal protective equipment: safety goggles, smock or apron (while loading gels and during electrophoresis), and protective gloves (during staining).

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