Shaffer Lab:Protocols

Shaffer Lab Home

Shaffer Lab Links

Lab Safety & General Guidelines

Please wear proper laboratory attire any time you are in the lab.

Food and drinks are not allowed in the lab. Please keep these in the break room.

Always wear gloves when doing benchwork. Remember to remove gloves before leaving the lab!

Properly dispose of all hazardous and non-hazardous waste. See section on "waste tags."

Make your own aliquots of stock solutions to avoid contamination.

Label and date everything, including new aliquots of water or cleaning solution.

Please turn off equipment and return all lab supplies when finished.

If we are running low on any reagents, notify the lab technician to order more.

Keep all personal belongings on or beneath desks and out of aisles.

Recipes for general-use buffers

50X Tris-acetate-EDTA (TAE) (makes 1L)
1. Obtain a bottle with at least 1L capacity
2. Measure out 242 g of solid Tris Base and add to bottle
3. Add 600 ml distilled water
4. Place beaker on stirring block, add stir bar
5. Stir until Tris is dissolved
6. Add 100 ml 0.5M EDTA
7. Add 57.1 ml Glacial acetic acid (wear eye protection and use caution)
8. Add water until total volume is 1L
9. Remove stir bar with magnet
10. Store at room tempurature

5M Sodium Chloride (NaCl) (makes 250mL)
1. Obtain a beaker with at least 250mL capacity
2. Measure out 73.05g of solid sodium chloride (NaCl) and add to beaker
3. Add distilled water up to total volume of slightly less than 250mL
4. Place beaker on stirring block, add stir bar
5. Stir and heat slightly
6. Add water until total volume is 250mL and sodium chloride is totally dissolved
7. Remove stir bar with magnet and place solution in a 500mL bottle with a loosely fitted cap
8. Autoclave (liquid cycle: ~20 mins)
9. Once cooled, run solution through sterile filter system

• Obtain a short length of plastic hose to connect vacuum line to filter unit
• Make sure receiving unit (bottom bottle) and filter unit (top half) are held together tightly
• Slowly turn on vacuum and keep at a low pressure
• Pour solution into filter unit and allow to drain into receiving unit
• Turn off vacuum, remove filter unit, screw on cap

10. Label container (common chemical name, concentration, date, your initials)

10X loading dye(makes 50mL)
1. Obtain a 50ml Falcon tube
2. Add 19.5mL glycerol
3. Add 1mL 0.5M EDTA
4. Add ~39mL distilled water top bring volume to 50mL
5. Close lid and shake
6. Transfer 10mL into 15mL Falcon tube
7. Measure 0.025g of Orange G (>100bp*), Bromophenol blue (400-500bp*), AND/OR Xylene cyanol (10,000-4000 bp*)

• based on 0.5-1.5% agarose gel

8. Add dye(s) to Falcon tube
9. Close lid and shake
10. Store in refrigerator
11. Label container (common chemical name, concentration, dye(s) added, date, your initials)

1M Tris(-Cl), pH 8 (makes 250mL)
1. Obtain a beaker with at least 250mL capacity
2. Measure out 30.275g of Tris base and add to beaker
3. Add 200mL distilled water
4. Place beaker on stirring block, add stir bar
5. Stir and heat slightly to dissolve Tris base
6. Calibrate pH meter with pH 7.0 buffer and pH 10.0 buffer
7. Measure pH of solution
8. Add 9mL of concentrated HCl (be very careful, this acid is highly caustic) and measure pH
9. Continue adding HCl dropwise until pH measures 8.0
10. Add distilled water to total volume of 250mL
11. Remove stir bar with magnet and place solution in a 500mL bottle with a loosely fitted cap
12. Autoclave (liquid cycle: ~20 mins)
13. Once cooled, run solution through sterile filter system

• Obtain a short length of plastic hose to connect vacuum line to filter unit
• Make sure receiving unit (bottom bottle) and filter unit (top half) are held together tightly
• Slowly turn on vacuum and keep at a low pressure
• Pour solution into filter unit and allow to drain into receiving unit
• Turn off vacuum, remove filter unit, screw on cap

14. Label container (common chemical name, concentration, date, your initials)

Top

Wayne Lab Home