Alberts, Bruce, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts and James D. Watson (1994). Molecular Biology of the Cell. New York; Garland Publishing, Inc.

Campbell, Neil A. (1996) Biology. Redwood City, CA; The Benjamin Cummings Publishing Company, Incorporated.

Curtis, Helena and N. Sue Barnes (1989) Biology. New York, NY; Worth Publishers, Inc.

Cooper, Necia Grant, ed. (1994) The Human Genome Project - Deciphering the Blueprint of Heredity. Mill Valley, CA; University Science Books.

Kreuzer, Helen and Adrianne Massey (1996) Recombinant DNA and Biotechnology. Washington, D.C.; ASM Press.

Micklos, David A. and Greg A. Freyer (1990) DNA Science. Cold Spring Harbor, NY; Carolina Biological Supply Company and Cold Spring Harbor Press.

Winternitz, Katherine A., et al. (1996) Biological Science:A Molecular Approach. BSCS Blue

Version, 7th ed. Lexington, MA; D.C. Heath and Co.

 

Appendix II. List of Suppliers

Products Purchased from Fisher Scientific

Appendix III. Kit Contents

Equipment and Supplies

For the class:

1 electric frying pan
2 water baths, each with 1 thermometer and 2 microtube racks, OR thermal cycler
1 power supply
2 sequencing electrophoresis apparati with 6% polyacrylamide denaturing gels
Boxes of flat microcapillary pipet tips
2 - 16" x 13" pieces of Whatman filter paper
1 - 16" x 13" Plexiglass plate
1 roll saran wrap
1 roll aluminum foil
1 roll parafilm
16 500 ml beaker
UV crosslinker
Boxes of disposable gloves
Mini-centrifuge
Extra disposable tips

1 class supply kit

2 P-200 automatic pipettors
1 biohazard bag
2.25" x 12" pieces of Hybond N+ hybridization transfer membrane (1 per group)

For each lab group:

1 lab supply kit, containing:

1 P-20 automatic pipettor and pipet tips
1 stopwatch
1 thermometer
1 felt marker
1 pencil
0.5 ml microfuge tubes, assorted colors
1 microtube rack
1 waste tip box
1 round tube rack
1 transfer pipet
1 metal spatula
1 forceps
1 graduated cylinder, 25 ml
1- 3" x 15" tray (Rubbermaid drawer organizer)

1. 1- 250 ml graduated cylinder
2. 1- distilled waterbottle
3. 1- plastic page protector

Solutions

Day 1  
A, C, G and T reaction mixes for thermal cycling protocol

(Store these six reagents in a –20°C freezer. Return to the freezer between classes. A few minutes at room temperature is permissible, but these reagents should not be left out overnight. )

1. A Reaction Mix

2. C Reaction Mix

41.7ul ddC Termination Mix (purple capped USB tubes)
4.5ul Concentrated Reaction Buffer (included in USB kit)
2.4ul 2.7uM Forward or Reverse biotin-tagged Sequencing Primer

3. G Reaction Mix

41.7ul ddG Termination Mix (purple capped USB tubes)
4.5ul Concentrated Reaction Buffer (included in USB kit)
2.4ul 2.7uM Forward or Reverse biotin-tagged Sequencing Primer

4. T Reaction Mix

41.7ul ddT Termination Mix (purple capped USB tubes)
4.5ul Concentrated Reaction Buffer (included in USB kit)
2.4ul 2.7uM Forward or Reverse biotin-tagged Sequencing Primer

0.11 pmole/µL DNA template

Before Day 1, teachers should note the volume of template received so that s/he can divide it evenly between groups. When possible, have at least 2 groups in each class use each template so that students can compare their results.

Included in US Biochemical kit.

Day 2

7. 5x TBE (alternate running buffer) For 1 L:

54 g Tris base 
27.5 g Boric acid 
20 ml 0.5M EDTA pH 8
H₂0 to 1L

8. 6% Acrylamide mix For 1 L:

150 mL 40% Acryl:Bis 19:1
200 ml 5x TBE
420 g Urea
distilled H2O to 1 L
(filter sterilized acrylamide mixtures and store in the dark at 4° C)

Day 3

9. Blocking Solution (store at room temperature) For 1 L:

7.3 g solid NaCl
62.5 ml 0.4M sodium phosphate, pH 7.2
50g SDS  (sodium dodecyl sulfate)
Distilled H₂0 to 1L

If SDS comes out of solution before use, place bottle in warm water bath for a few minutes until it goes back in solution.

10. 0.4 M Monobasic Sodium Phosphate, pH 7.2 For I L:

Monobasic sodium phosphate 55.29 g
Distilled H₂0 to 1L

11. 0.4 M Dibasic Sodium Phosphate, pH 7.2 For 1 L:

Dibasic sodium phosphate 107.29 g
Distilled H₂0 to 1L

To make sodium phosphate at pH 7.2, make 0.4 M stocks of mono- and di-basic sodium phosphate. Starting with about 600 ml of the dibasic stock, add monobasic until the pH reaches 7.2. There are charts to calculate the amount of each salt to add to achieve a given pH, but I find this method more practical.

12. Buffer S (for dissolving streptavidin) For 100 ml:

10 ml 0.1 M sodium phosphate, pH 7.2
3.75 ml 4 M NaCl
Distilled H₂0 to 1L

13. 1X Wash Solution I (store at room temperature) For 1 L:

Blocking solution 100ml
Distilled H₂0 to 1L

(1:10 dilution blocking solution in distilled water.)

If SDS comes out of solution before use, place bottle in warm water bath for a few minutes until it goes back in solution.

14. 10X Wash Solution II (store at room temperature) For 1 L:

100 ml 1 M Tris-HCl, pH 9.5
25 ml 4 M NaCl
10 ml 1 M MgCl₂
Distilled H₂0 to 1L

15. 1x Wash Solution II (store at room temperature) For 1 L:

100ml 10x Wash solution II
Distilled H₂0 to 1L

16. Streptavidin at 1 mg/ml (store at 4°C)

(Alkaline Phosphatase) at 0.5 mg/ml (store at 4°C)

Note that New England Biolabs provides biotin-tagged AP at 0.38 mg/ml.

100 mg/ml nitro blue tetrazolium chloride in 70% dimethylformamide

50 mg/ml 5-bromo-4-choloro-3-indolyl-phosphate, 4-toluidine salt in dimethylformamide

20. Color Substrate Buffer (store at room temp.) For 1 L:

100 ml 1 M Tris-HCl, pH 9.5
25 ml 4 M NaCl
50 ml 1 M MgCl₂
10 ml 10 mM ZnCl₂
Distilled H₂0 to 1L

21. Stop Solution (store at room temperature) For 1 L:

10 ml 1 M Tris-HCl, pH 8
2 ml 0.5 M EDTA
Distilled H₂0 to 1L