Reagent Recipes

Reagent Information

Reagent	Abbreviation	Stock Solution Concentration	Solvent	Stock Concentration to Working Concentration	Notes
Bromo-chloro-indolyl-galactopyranoside	X-gal	20mg/ml	Dimethylformamide (DMF)	250x	Light sensitive. Use DMF in a chemical hood; pre-made DMF aliquots are available in the "Molecular Biology" box in the -20 ºC freezer.
Isopropyl β-D-1-thiogalactopyranoside	IPTG	0.1M	H2O	1000x	Light sensitive. Use at 1mM in S. pneumoniae with pLac; stock is a 100x solution for this purpose. Molecular weight: 238.31 g/mol
Catalase	Cat	30,000U / ml	PBS	100x	Light sensitive. Do not vortex when dissolving.
Triphenyl tetrazolium chloride	TTC	50mg / ml	H2O	1000x
NAD(+) / Factor V	NAD(+)	1mg / ml	H2O	66.67x [15ug/ml]	Light sensitive.
Hemin / Factor X	Hemin	2mg / ml	H2O	133.33x [15ug/ml]	Light sensitive.
Ethylhydrocupreine hydrochloride	Optochin	5mg/ml	H20	1000x	Light sensitive.
Lysozyme	Lysozyme	10mg /ml	H2O

Glycerol for freezing aliquots

We use sterile 80% glycerol in water. We dilute this 1:3 (400ul 80% glycerol; 1200ul culture) for creating freezer stocks with a final glycerol concentration of 20%.

MgCl₂

When working with MgCl₂ be aware that when added to water a significant exothermal reaction occurs.

10x PBS

Used to create 1x PBS for dilution or washing cells of any species.

For 1 liter:

• NaCl 80g
• KCl 2g
• Na₂HPO₄ 14.4g
• KH₂PO₄ 2.4g

Stir salts with MilliQ water. This may take a significant time to dissolve!

Adjust pH with NaOH to 7.4. This will take a significant amount of NaOH; add in 200ul increments.

Tris Buffer

For Tris buffer at pH 8.0 (at 25 degrees)

• 4.44 g/L Tris HCl
• 2.65 g/L Tris Base

Do not adjust pH or check the pH with the MEE lab pH probe. This will destroy the probe!

Catalase

1. Our catalase is 13,000U/mg. We want a final concentration of 30,000U / ml. Therefore, to make 10ml, we need to use 23.07mg, or 0.0231g. To make 40ml, use 0.0924g.
2. The catalase is in the chemical box on a lower shelf of our -20. There are 2 brands: Fisher and MP Chemical. Only use the MP Chemical brand.
3. Use the precise balance of Karl's lab to measure out the catalase. Use a folded piece of weighing paper instead of a weighboat. Catalase is quite sticky — be prepared for this!
4. Put catalase into a 50ml sterile plastic tube.
5. Pipet in PBS, measuring out the volume using a serological pipet. Normally, we would use 10ml or 40ml.
6. Gently shake until dissolved. DO NOT VORTEX.
7. Sterilely filter solution using a syringe filter and place into a fresh, sterile plastic tube.
8. Label top with contents and date; limit exposure to light.
9. Keep in refrigerator; re-suspended catalase cannot be stored at -20 degrees.

Protein Purification Lysis Buffer

• Composition:
  • 50 mM sodium phosphate buffer (pH 7.6)
  • 10 mM imidazole
  • 300 mM NaCl

1. To make 50 mM sodium phosphate buffer (pH 7.6):
   1. Add 42.25 mL of 1 M Na2HPO4 and 7.75 mL of 1 M NaH2PO4 to 1 L Erlenmeyer flask.
2. To make 10 mM imidazole:
   1. Add 10 mL of 1 M imidazole to flask.
3. To make 300 mM NaCl:
   1. Add 60 mL of 5 M NaCl to flask.
4. Fill flask up to 1 L with MilliQ H2O.
5. Adjust pH to 7.6.
6. Sterilize the lysis buffer in the autoclave.

Protein Purification Wash Buffer

• Composition:
  • 50 mM sodium phosphate buffer (pH 7.6)
  • 30 mM imidazole
  • 300 mM NaCl

1. To make 50 mM sodium phosphate buffer (pH 7.6):
   1. Add 42.25 mL of 1M Na2HPO4 to a 1L Erlenmeyer flask.
   2. Add 7.75 mL of 1M NaH2PO4 to the 1L Erlenmeyer flask.
2. To make 30 mM imidazole:
   1. Add 30 mL of 1M imidazole to the 1L Erlenmeyer flask.
3. To add 300 mM NaCl:
   1. Add 60 mL of 5M NaCl to the 1L Erlenmeyer flask.
4. Fill flask to 1 L with Milli Q H2O.
5. Adjust pH to 7.6.
6. Sterilize the wash buffer in the autoclave.

Protein Purification Elute Buffer

• Composition:
  • 50 mM sodium phosphate buffer (pH 7.6)
  • 250 mM imidazole
  • 100 mM NaCl

1. To make 50 mM sodium phosphate buffer (pH 7.6):
   1. Add 42.25 mL of 1 M Na2HPO4 and 7.75 mL of 1 M NaH2PO4 to 1 L Erlenmeyer flask.
2. To make 250 mM imidazole:
   1. Add 250 mL of 1 M imidazole to flask.
3. To make 100 mM NaCl:
   1. Add 20 mL of 5 M NaCl to flask.
4. Fill flask up to 1 L with MilliQ H2O.
5. Adjust pH to 7.6.
6. Sterilize the elute buffer in the autoclave.

Stripping Buffer

• Composition:
  • 20 mM sodium phosphate buffer (pH 7.6)
  • 50 mM EDTA (pH 7)
  • 0.5 M NaCl

1. To make 20 mM sodium phosphate buffer (pH 7.6):
   1. Add 16.9 mL of 1M Na2HPO4 to a 1L Erlenmeyer flask.
   2. Add 3.1 mL of 1M NaH2PO4 to flask.
2. To make 50 mM EDTA (pH 7):
   1. Add 100 mL of 0.5M EDTA to flask. If solid, add 18.6 g of EDTA (MW 372).
3. To make 0.5 M NaCl:
   1. Add 100 mL of 5M NaCl to flask.
4. Fill flask to 1 L with MilliQ H2O
5. Adjust pH to 7
6. Sterilize the stripping buffer in the autoclave.