Reagent Recipes: Difference between revisions
Jump to navigation
Jump to search
PreDec2022>Dcapcha No edit summary |
PreDec2022>Dcapcha No edit summary |
||
Line 102: | Line 102: | ||
==Lysis Buffer== | ==Lysis Buffer== | ||
* Composed of: 50 mM sodium phosphate buffer pH 7.6, 10 mM imidazole, 300 mM NaCl | |||
# Make 50 mM sodium phosphate buffer pH 7.6 | # Make 50 mM sodium phosphate buffer pH 7.6 | ||
## Add 42.25 mL of 1 M Na2HPO4 and 7.75 mL of 1 M NaH2PO4 | ## Add 42.25 mL of 1 M Na2HPO4 and 7.75 mL of 1 M NaH2PO4 | ||
Line 112: | Line 112: | ||
# Adjust pH to 7.6 | # Adjust pH to 7.6 | ||
# Sterilize | # Sterilize | ||
==Wash Buffer== | |||
==Elute Buffer== | |||
* Composed of | |||
** 50 mM sodium phosphate buffer pH 7.6 |
Revision as of 15:25, 3 June 2021
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 | Use DMF in a chemical hood |
Isopropyl β-D-1-thiogalactopyranoside | IPTG | 0.1M | H2O | 1000x | Light sensitive. 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. |
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
- 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.
- Use the precise balance of Karl's lab to measure out the catalase. Use a folded piece of weighing paper instead of a weighboat.
- Put catalase into a 15ml sterile plastic tube.
- Pipet in PBS, measuring out the volume using a serological pipet. Normally, we would use 10ml.
- Gently shake until dissolved. DO NOT VORTEX.
- Sterilely filter solution using a syringe filter and place into a fresh, sterile plastic tube.
- Label top with contents and date; limit exposure to light by creating an aluminum foil sleeve.
- Keep in refrigerator
Lysis Buffer
- Composed of: 50 mM sodium phosphate buffer pH 7.6, 10 mM imidazole, 300 mM NaCl
- Make 50 mM sodium phosphate buffer pH 7.6
- Add 42.25 mL of 1 M Na2HPO4 and 7.75 mL of 1 M NaH2PO4
- Make 10 mM imidazole
- Add 10 mL of 1 M imidazole
- Make 300 mM NaCl
- Add 60 mL of 5 M NaCl
- Fill up to 1 L with MilliQ H2O
- Adjust pH to 7.6
- Sterilize
Wash Buffer
Elute Buffer
- Composed of
- 50 mM sodium phosphate buffer pH 7.6