| Ingredient | Amount | Final Concentration |
|---|---|---|
| Trisma base | 12.1 g | 0.1 M |
| KCl | 59.7 g | 0.8 M |
| EDTA | 37.2 g | 0.1M |
| Spermidine-HCL | 2.55 g | 10 mM |
| Spermine-HCL | 3.48 g | 10 mM |
Adjustment: pH to 9.4-9.5 with NaOH. Store at 4 °C.
| Ingredient | Amount | Final Concentration |
|---|---|---|
| 10X HB stock | 100 ml | 1X |
| Sucrose | 171.15 g | 0.5M |
Store at 4 °C.
Adjustment: Before use add beta-mercaptoethanol to 0.15%.
| Ingredient | Amount | Final Concentration |
|---|---|---|
| 10X HB stock | 10 ml | 1X |
| Sucrose | 17.12 g | 0.5M |
| Triton X-100 | 20 ml | 20% |
Store at 4 °C.
| Ingredient | Amount | Final Concentration |
|---|---|---|
| 10X HB stock | 100 ml | 1X |
| Sucrose | 171.15 g | 0.5M |
| Triton X-100 (20% stock in 1 X HB) | 25 ml | 0.5% |
Store at 4 °C. Adjustment: Before use add beta-mercaptoethanol to 0.15%.
| Ingredient | Amount | Final Concentration |
|---|---|---|
| Na lauryl sarcosine | 20g | 2% |
Adjustment: Autoclave
| Ingredient | Amount | Final Concentration |
|---|---|---|
| EDTA | 500 ml of 1 M stock | 0.5M (pH 9.0-9.3) |
| Na lauryl sarcosine | 500 ml of 2% stock | 1%< |
Adjustment: Before use, add Proteinase K to 0.1 mg/ml
| Ingredient | Amount | Final Concentration |
|---|---|---|
| EDTA | 186.1 g | 0.5 M |
| NaOH | 20g |
Adjustment: pH to 8.0 with NaOH or HCl. Autoclave.
| Ingredient | Amount | Final Concentratio |
|---|---|---|
| EDTA-di sodium salt | 362.2 g | 1.0 M |
| NaOH | 40g |
Adjustment: pH to 9.0 to 9.3 with NaOH. Autoclave.
| Ingredient | Amount | Final Concentration |
|---|---|---|
| Trisma base | 121.1 g | 1M |
| HCl | 42 ml | |
| ddH20 | 800 ml |
Adjustment: pH to 8.0 with HCl and adjust volume to 1 liter. Autoclave.
| Ingredient | Amount | Final Concentration |
|---|---|---|
| Tris-HCl | 10 ml of 1 M stock, pH 8.0 | 10 mM |
| EDTA | 2 ml of 0.5 M stock, pH 8.0 | 1.0 mM |
Adjustment: Autoclave
: PMSF is a highly toxic acetylcholinesterase inhibitor. To avoid possible lab contamination, we purchase small 250 mg aliquots and then add isopropanol as above to the entire bottle to make a final concentration of 100 mM.
| Ingredient | Amount | Final Concentration |
|---|---|---|
| PMSF | 250 mg vial | 100 mM |
| Isopropanol | 14.35 ml |
Store at 4 °C.
| Ingredient | Amount | Final Concentration |
|---|---|---|
| Bacto tryptone | 20g | 2% |
| Bacto yeast extract | 5g | 0.5% |
| NaCl | 10 ml (1 M stock) | 10 mM |
| KCl | 2.5 ml (1 M stock) | 2.5 mM |
| ddH2O | up to 980 ml | - |
Adjustment: Adjust pH to 7.0, autoclave, and cool to room temperature and then add.
| MgSO4/ MgCl2 Stocka | 10 ml (2 M stock) | 20 mM |
| Glucoseb | 10 ml (2 M stock) | 20 mM |
a: Prepare and filter-sterilize a 2 M Mg++ stock ( 1 M MgCl2 6H
2O + 1 M MgSO4 - 7 H
2O)
b: Prepare and filter-sterilize a 2 M Glucose stock.
| Ingredient | Amount | Final Concentration |
|---|---|---|
| Bacto tryptone | 10g | 1% |
| Bacto yeast extract | 5g | 0.5% |
| NaCl | 10g | 1% |
| Agar (for plates) | 15g | 1.5% |
Adjustment: Adjust pH to 7.5. Autoclave.
| Ingredient | Amount | Final Concentration |
|---|---|---|
| Glucose | 0.9g | 50 mM |
| EDTA (pH 8.0) | 2 ml of 0.5 M stock | 10 mM |
| Tris-HCl (pH 8.0) | 5 ml of 1 M stock | 25 mM |
| Lysozyme | 5 mg/ml |
Adjustment: Adjust volume to 100 ml with pure water and autoclave. Store at 4 °C. Add lysozyme just before use.
| Ingredient | Amount | Final Concentration |
|---|---|---|
| NaOH | 1/2 volume of 0.4 N stock (8g/500 ml) | 0.2 N |
| SDS | 1/2 volume of 2% stock (10g/500ml) | 1% |
Adjustment: Mix NaOH and SDS stocks just prior to use.
| Ingredient | Amount | Final Concentration |
|---|---|---|
| Potassium Acetate | 60 ml of 5 M KOAc | |
| Glacial Acetic Acid | 28.5 ml | |
| H2O | 11.5 ml |
| Ingredient | Amount | Final Concentration |
|---|---|---|
| NaCl | 175.3 g/l | 3.0 M |
| Na Citrate | 88.2 g/l | 3.3 M |
Adjustment: Dissolve in pure water and adjust pH to 7.0 with Citric acid.
| Ingredient | Amount | Final Concentration |
|---|---|---|
| Trisma Base | 242 g | |
| Glacial acetic acid | 57.1 ml | 2.5 M Tris-Acetate |
| EDTA | 100 ml of 0.5 M EDTA (pH 8.0) | 50 mM |
Dilute 50 fold with pure autoclaved water.
| Ingredient | Amount | Final Concentration |
|---|---|---|
| Trisma Base | 54 g | 0.45 M |
| Boric Acid | 27.5 g | 0.45 M |
| EDTA | 20 ml (0.5 M stock) | 10 mM |
The pH should be about 8.3 and should not require adjustment.
Mode of action: A bacteriostatic agent that interferes with bacterial protein synthesis by binding to the 50S subunit of ribosomes and preventing peptide bond formation.
Mechanism of resistance: The resistance gene specifies an acetylytransferase that acetylates and thereby activates the antibiotic.
Stock solution: 50 mg/ml in 100% ethanol. Store at -20 °C.
pBeloBAC11 plasmid preparation: 30 ug/ml
BAC transformation & minipreps: 12.5 ug/ml
Stock solution: 200 mg/ml in H2O. Filter sterilize and store at -20 °C.
Stock solution: 20 mg/ml in NN-dimethyl formamide. Store at -20 °C.
BAC transformation plates: 240 ml/plate (15 X 150 mm)
| Ingredient | Amount | Final Concentration |
|---|---|---|
| Glycerol | 350ml | 35% |
| 5X TBE | 10ml | 1X |
| 0.5M EDTA (pH 8.0) | 20ml | 20mM |
| 20% SDS | 5ml | 0.2% |
| 10% bromophenol blue | 30ml | 0.6% |
| ddH2O | 85ml | - |
首页
