Lab Reagent Prep

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First Thing

In the following lists, you can find a number of lab reagents with stock concentration (SC), and working concentration (WC). Be sure, when making stock or working concentration reagents, you understand the solubility of your reagent, and what is the appropriate solvent. Also be sure to understand what type of container you should keep your reagent in. If the reagent needs to be in glass, or perhaps kept out of light, make sure you choose your storage container wisely.

Lab Reagents - Stock solutions

• Potassium Tetrathionate ~ 302 g/mol (powder should be stored at 4C)

- Dissolve powder in room temperature distilled H2O, to a maximum concentration of 300mM (~0.09g/mL). Make fresh Potassium tetrathionate each time you plan to use it, filter sterilize, and only add to media that is < 55C. Working concentration for most uses in the Bucci lab is 1mM - 2mM.

• 2,2'-Bipyridyl - ~156 g/mol (powder stored at room temp). Often abbreviated "DP"

- Dissolve powder in room temperature 100% Ethanol (EtOH), to a concentration of 200mM (~31 mg/mL), which is 1000x. Make fresh DP each time you plan to use it. Does not need to be filter sterilized. Do not autoclave. Add to media that has cooled. Working concentration is 0.2mM

• X-Gal

- SC: 20mg/mL, which is 1000x above the WC of 20ug/ml. Solvent is DMSO! Prepare by the mL in Eppendorff tubes (Black) or wrap with foil to keep out of the light, and store in the -20C freezer.

• Kanamycin Sulfate

- SC: 50mg/mL in DI H2O. Filter sterilize. WC is 100ug/mL. Store the SC at 4C.

• Carbenicillin

- SC: 50mg/mL in HI H2O. Filter sterilize. WC is 100ug/mL. Store the SC at -20C.

• Chloremphenicol

- SC: 25mg/mL in 100% EtOH. No need to filter sterilize. WC is 25ug/mL. Store SC at -20C.

Lab Reagents - Buffers

• Column Buffer (Amylose Resin chromatography)

200mM NaCl (11 g/L), 20mM Tris-HCl (3.14g/L), 1mM EDTA (0.372g/L). Note: We have EDTA Disodium Salt Dihydrate with a MW = 372.24. Therefore 1mM EDTA is 0.372g/L of the EDTA Disodium Salt Dihydrate. If using a different powder, please convert accordingly. Note: EDTA is very difficult to dissolve at pH < 7.5.

• Column Buffer - No EDTA (Amylose Resin chromatography)

200mM NaCl (11 g/L), 20mM Tris-HCl (3.14g/L)

• Elution Buffer (Amylose Resin chromatography)

200mM NaCl (11 g/L), 20mM Tris-HCl (3.14g/L), 10mM Maltose (3.42g/L). Filter sterilize after mixing. Note: We have Maltose Monohydrate with a MW of 342.3. Therefore 10mM Maltose is 3.42g/L of the Maltose Monohydrate. If using a different powder, please convert accordingly.

Lab Reagents - Media

• Microcin Production media:

10 g/L Peptone, 1x M9 Salts, 0.1mM CaCl2, 2mM MgSO4, 0.2mM DP, and usually antibiotic and inducing/repressing molecule (L-arabinose/Glucose/IPTG/aTc, etc) as needed. It is recommended to make stock solutions of the Peptone at 20g/L, 5x M9, 1M CaCl2, and 1M MgSO4 and then mix components together as needed. DP and antibiotic should always be added fresh.

• CAS agar plates:

Simplified

Rhodococcus-related Media

• Colloidal Chitin