1. Calculate total amount of volume of LB agar you would need to use
   • You need about 25mL for every 10cm plate and 10mL for every 6cm plate. (e.g. To make 20 x 10cm plates, you need 500mL of LB.)
2. For every 100mL of LB agar needed, measure out 4g of LB Agar Powder.
   • e.g. if you were to make 500mL of LB, you need 20g of LB Agar Powder
3. Put the agar powder in a flask
   • Note: Use a much bigger flask to prevent overflow when autoclaiving.
   • e.g. If you are making 500mL of LB, use a 750mL or 1L flask.
4. Put deionized water into the flask up to the desired volume.
   • Note: Do not mix. LB Agar is not supposed to dissolve in room temperature. If you mix the powder it will get stuck on the walls of the flask
5. Autoclave.
6. After autoclave, move to the hood. Let the LB agar cool until it’s less than 60ºC or until you can touch the flask. While waiting, you can start labeling the plates.
7. Insert any antibiotics/sugars needed. If you are not sure about the ratio, you can follow this simple example:

   50mL LB + 500µL Arabinose* + 50µL Ampicillin**
   *2mg/mL of Arabinose Stock Solution
   **100mg/mL of Ampicillin Stock Solution

8. Swirl the antibiotics/sugars that you just added for a minute.
9. Plate the LB agar into the petri dishes until it is about ⅓ of the level. Do not forget to keep the lid on before and after you plate.
10. Let it cool for about 5 minutes.
11. Invert each plate so that it is upside down
12. Store in the 4ºC freezer

This is how you make LB Broth.

Materials

• 0.5% (w/v) yeast extract
• 2% (w/v) tryptone
• 10 mM NaCl
• 2.5 mM KCl
• 20 mM MgSO4

Per liter:

• 5 g yeast extract
• 20 g tryptone
• 0.584 g NaCl
• 0.186 g KCl
• 2.4 g MgSO4

Note: Some formulations of SOB use 10 mM MgCl2 and 10 mM MgSO4 instead of 20 mM MgSO4. SOB medium is also available dry premixed from Difco, 0443-17.
Adjust to pH 7.5 prior to use. This requires approximately 25 ml of 1M NaOH per liter.

15/10 medium

Growth of competent TOP10 cells in Example 2 of the Bloom05 patent is performed in 15/10 broth, which is similar to SOB:

• 1.5% yeast extract
• 1% Bacto-Tryptone
• 10mM NaCl
• 2mM KCl
• 10 mM MgCl2
• 10 mM MgSO4

General Overview:

1. D-Luciferin is too large of a chemical to cross the plasma membrane of e. Coli so cell lysis is required to extract luciferase.
2. After cell lysis, the reagent solution can be added to the lysis buffer. Light should be emitted within 5 to 10 seconds of adding the reagent solution.
3. The luciferase/luciferin reaction at 22.5 ºC theoretically offers the greatest light intensity.
4. Solutions of D-Luciferin should be aliquotted and stored in darkness at -80 ºC

Materials:

• D-Luciferin free acid
• ATP
• MgSO4 · 7H2O
• 1M HEPES Buffer
• Lysozyme
• 10 mM Tris-HCl

Lysis Buffer:

For E. coli cell lysis, use a freshly prepared lysozyme solution (10 mg/ml) in 10 mM Tris-HCl, pH 8.0.

Reagent Solution:

Prepare using ATP free water. Combine 1 mM luciferin or luciferin salt, 3 mM ATP and 15 mM MgSO4 in 30mM HEPES buffer, pH 7.8. Store substrate solution at -20ºC in polypropylene or glass.

Preparing 1mM D-Luciferin:

Directions for a 5 mg sample: Dissolve 0.034 gr dithiothreitol in 22 mL of QH2O. Add 0.2 mL of this DTT solution to the 5 mg d-luciferin. Add 4 uL of 10M NaOH to dissolve the luciferin. Dilute this into the remaining 21.8 mL of DTT solution and store as aliquots at - 80 ºC in darkness until use.

Procedure:
Bacterial lysis:

1. After 12-18 hours of inoculation of bacteria expressing luciferase plasmid, pipette 2 mL of cell culture into a clean 2 mL tube. Centrifuge at 10,000 rpm for 1 minute. Pour out liquid into a collection beaker. Continue this process until all of the cell culture (in the inoculation tube) is gone.
2. Resuspend the pellets in 350 ml of STET buffer (10 mM Tris-HCl with 1 mM EDTA)
3. Add 25 uL - 30 uL of lysozyme buffer to the resuspended pellet.
4. Mix by vortexing for 3 seconds.
5. Incubate for 2 hours at room temperature.
6. If the reagent is not added immediately, store the lysed bacteria in the -20 ºC freezer until use.

Addition of Reagent Solution:

1. Following the above instructions, prepare a 1mM sample of D-Luciferin.
2. Following the above recipe, prepare the reagent solution.
3. In a dark room, add about 250-350 uL of reagent solution to each sample of lysis product.
4. Light should be emitted within two-three seconds.

Example Calculations:
Lysis Buffer (Desired Total Volume: 15mL)

Chemical Name:	Tris-HCl	EDTA	NaCl
Molecular Weight:	N/A	292.23 g/mol	58.44 g/mol
Molarity Desired:	10 mM	1mM	0.1M
Calculation:	Dilute 1M Tris-HCl:
Final Amount:	150 uL (+14.85 mL ddH2O)	0.00438 g	0.08766 g

Lysozyme Solution (Desired Total Volume: 15mL)

Lysozyme Solubility:	10 mg lysozyme in 1 mL of 10 mM Tris-HCl
Desired Amount of Lysozyme Solution to Make:	15 mL
Amount of Lysozyme Needed:	10 mg x 15 = 150 mg
Amount of 10 mM Tris-HCl Needed:	15 mL

Reagent Solution (Desired total volume: 22 mL)

Chemical Name:	ATP disodium salt trihydrate	MgSO4•7H2O	HEPES Buffer	D-Luciferin free acid
Molecular Weight:	605.24 g/mol	246.5 g/mol	238.3 g/mol	280.33 g/mol
Molarity Desired:	3 mM	15 mM	30 mM	1 mM
Calculation:				Use the 1 mM stock solution created earlier
Final Amount:	0.039945 g	0.081345 g	0.1573 g	22 mL

This is the conditions we used to express chromoproteins.