Griffitts:Stock solutions: Difference between revisions
Line 266: | Line 266: | ||
Sterile filter | Sterile filter | ||
===10% [[SDS]] (75 mL)=== | |||
* 75 mL ddH<sub>2</sub>O | |||
* 7.5 g Sodium dodecyl sulfate ([[SDS]]) | |||
Do '''not''' sterilize | |||
===20% [[SDS]] (75 mL)=== | ===20% [[SDS]] (75 mL)=== | ||
* 75 mL ddH<sub>2</sub>O | * 75 mL ddH<sub>2</sub>O |
Revision as of 13:19, 5 March 2010
Antibiotics
Ampicillin (40 mL)
- 1 g Ampicillin
- 40 mL ddH2O
- Filter sterilize
- Store in refrigerator
Chloramphenicol (40 mL)
- 0.3 g Chloramphenicol
- 40 mL 50% Ethanol
- Do not filter sterilize
- Store in refrigerator
Erythromycin (20 mL)
- 1.0 g Erythromycin
- 20 mL 100% ethanol (in fume hood)
- Do not filter sterilize—the ethanol may dissolve the filter
- Store in the -20°C Freezer
Gentamicin (40 mL)
- 0.3 g Gentamicin
- 40 mL ddH2O
- Filter sterilize
- Store in refrigerator
Hygromycin (40 mL)
Kanamycin (40 mL)
- 0.3 g Kanamycin
- 40 mL ddH2O
- Filter sterilize
- Store in refrigerator
Nalidixic acid (30 mL)
- 0.9 g Nalidixic Acid
- 0.36 g NaOH
- 30 mL ddH2O
- Filter sterilize
- Store in refrigerator
Neomycin (40 mL)
- 0.8 g Neomycin
- 40 mL ddH2O
- Filter sterilize
- Store in refrigerator
Rifampicin (20 mL)
- 1.0 g Rifampicin
- 20 mL N,N-dimethylformamide (DMF; in fume hood)
- Do not filter sterilize—the DMF will dissolve the filter
- Store in the -20°C Freezer
Spectinomycin (40 mL)
- 0.8 g Spectinomycin
- 40 mL ddH2O
- Filter sterilize
- Store in refrigerator
Streptomycin (40 mL)
- 2.0 g Streptomycin
- 40 mL ddH2O
- Filter sterilize
- Store in refrigerator
Tetracycline (40 mL)
- 0.1 g Tetracycline
- 40 mL 50% Ethanol
- Do not filter sterilize
- Store in refrigerator
Antibiotics Table
Antibiotic | Abbr. | Stripe | Stock | E. coli | S. meliloti | Solvent | Storage |
---|---|---|---|---|---|---|---|
Ampicillin | Ap | BLACK | 25 mg/mL | 100 μg/mL (250X) | N/A | ddH2O | 4°C |
Chloramphenicol | Cm | BROWN | 7.5 mg/mL | 30 μg/mL (250X) | N/A | EtOH | 4°C |
Erythromycin | Em | (none) | 50 mg/mL | N/A | N/A | EtOH | –20°C |
Gentamycin | Gm | PURPLE | 7.5 mg/mL | 3 μg/mL (2500X) | 15 μg/mL (500X) | ddH2O | 4°C |
Hygromycin | Hy | (none) | 5 mg/mL | 20 μg/mL (250X) | 20 μg/mL (250X) | ddH2O | 4°C |
Kanamycin | Km | YELLOW | 7.5 mg/mL | 30 μg/mL (250X) | N/A | ddH2O | 4°C |
Nalidixic acid | Na | (none) | 5 mg/mL | 30 μg/mL (1000X) | N/A | ddH2O | 4°C |
Neomycin | Nm | GREEN | 20 mg/mL | N/A | 80 μg/mL (250X) | ddH2O | 4°C |
Rifampicin | Rm | (none) | 50 mg/mL | N/A | N/A | DMF | –20°C |
Spectinomycin | Sp | ORANGE | 20 mg/mL | 80 μg/mL (250X) | 80 μg/mL (250X) | ddH2O | 4°C |
Streptomycin | Sm | BLUE | 50 mg/mL | N/A | 200 μg/mL (250X) | ddH2O | 4°C |
Tetracycline | Tc | RED | 2.5 mg/mL | 10 μg/mL (250X) | 10 μg/mL (250X) | EtOH | 4°C |
Triclosan | (none) | N/A | N/A | EtOH | 4°C |
Note: These parameters are intended to be compatible with pre-made 250-mL amounts of LB agar, such that usually 1 mL of stock solution is diluted into the LB agar. For liquid LB (typically 75 mL amounts) 0.3 mL of the stock solution will usually be added. Exception: Gentamycin for E. coli.
DNA
10 mM dNTPs
- Thaw and mix (stir) 100 mM stock solutions of dGTP, dATP, dTTP, dCTP (NEB; in freezer)
- Combine in a 1.5-mL tube:
Note: Store in the -20°C freezer in the box marked "Other Enzymes"
10X Taq buffer
- Label tubes: 10X Taq buffer
- Thaw and mix (stir) "Thermo pol buffer" (NEB; in freezer)
- Aliquot 375 μL of "Thermo pol buffer" into tubes—do not dilute!
Note: Store in the -20°C freezer in the box marked "Other Enzymes"
10X Pfx buffer
- Label tubes: 10X Pfx buffer
- Thaw and mix (stir) "Pfx50 10X PCR Buffer" (NEB; in freezer)
- Aliquot 375 μL of "Pfx50 10X PCR Buffer" into tubes—do not dilute!
Note: Store in the -20°C freezer in the box marked "Other Enzymes"
8X DNA loading
- 40% glycerol
- 1/50 dilution of 2% bromophenol blue
DNA ladder
- 147 μL ddH20
- 34 μL 8X DNA loading buffer
- 20 μL 1 kb ladder from NEB
- 2 μL 0.25 M EDTA
Note: use 5 μL per lane; 3kb band = 60 ng
Ethidium Bromide (10 mL)
- 10 mL ddH20
- 10 mg ethidium bromide
Note: use 8 μL per 50 mL gel
Media Additives
1 M Calcium Chloride (75 mL)
- 75 mL ddH2O
- 11.027 g calcium chloride dihydrate (CaCl2·2H2O)
Autoclave
2 M Magnesium Chloride (75 mL)
- 75 mL ddH2O
- 30.4965 g magnesium chloride hexahydrate (MgCl2·6H2O)
Autoclave
1 M Sodium Citrate (40 mL)
- 40 mL ddH2O
- 11.76 g sodium citrate tribasic dihydrate (Na3C6H5O7·2H2O)
- pH to 5.7 with HCl
Sterile filter
2% X-gal
- 10 mL N,N-dimethylformamide (DMF)(in fume hood)
- 0.2 g 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-gal)(in freezer)
Store at -20°C
0.2 M IPTG
- 5 mL ddH2O
- 0.238g isopropyl-β-D-thiogalactopyranoside (IPTG)(in freezer)
Sterile filter, aliquot, and store at -20°C
1 M Copper (II) sulfate (50 mL)
- 50 mL ddH2O
- 7.98 g CuSO4
Sterile filter
10% SDS (75 mL)
- 75 mL ddH2O
- 7.5 g Sodium dodecyl sulfate (SDS)
Do not sterilize
20% SDS (75 mL)
- 75 mL ddH2O
- 15 g Sodium dodecyl sulfate (SDS)
Do not sterilize
30% Acrylamide (75 mL)
- 75 mL ddH2O
- 22.5 g acrylamide
40% Acrylamide (75 mL)
- 75 mL ddH2O
- 30 g acrylamide
10% Ammonium persulfate (75 mL)
- 75 mL ddH2O
- 7.5 g ammonium persulfate (APS)
5 M NaCl (75 mL)
- 75 mL ddH2O
- 21.92 g NaCl
Autoclave
80% Glycerol (75 mL)
- 15 mL ddH2O
- 60 mL glycerol
Microwave for 30-second intervals on high until dissolved
Autoclave
1 M DTT (5 mL)
- 5 mL ddH2O
- 771 mg Dithiothreitol (DTT; in fridge)
Aliquot 500 μL into Eppendorf tubes Store in -20°C
Orcinol spray
- 5 mL ddH2O
- 25 mL methanol (in fume hood)
- 10 mL sulfuric acid (H2SO4 (in fume hood—be careful!)
- 200 mg orcinol