Griffitts:Stock solutions: Difference between revisions
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* 20 mL 100% ethanol (in fume hood) | * 20 mL 100% ethanol (in fume hood) | ||
* Do '''not''' filter sterilize—the ethanol may dissolve the filter | * Do '''not''' filter sterilize—the ethanol may dissolve the filter | ||
** Store in the | ** Store in the –20°C Freezer | ||
===Gentamicin (40 mL)=== | ===Gentamicin (40 mL)=== | ||
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* 20 mL N,N-dimethylformamide (DMF; in fume hood) | * 20 mL N,N-dimethylformamide (DMF; in fume hood) | ||
* Do '''not''' filter sterilize—the DMF will dissolve the filter | * Do '''not''' filter sterilize—the DMF will dissolve the filter | ||
** Store in the | ** Store in the –20°C Freezer | ||
===Spectinomycin (40 mL)=== | ===Spectinomycin (40 mL)=== | ||
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** 10 μl [[Wikipedia:Cytosine|dCTP]] | ** 10 μl [[Wikipedia:Cytosine|dCTP]] | ||
* Dispense 100-μL aliquots into 1.7-mL tubes | * Dispense 100-μL aliquots into 1.7-mL tubes | ||
Note: Store in the | Note: Store in the –20°C freezer in the box marked "Other Enzymes" | ||
===10X ''Taq'' buffer=== | ===10X ''Taq'' buffer=== | ||
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* Thaw and mix (stir) "Thermo pol buffer" (NEB; in freezer) | * Thaw and mix (stir) "Thermo pol buffer" (NEB; in freezer) | ||
* Aliquot 375 μL of "Thermo pol buffer" into tubes—do '''not''' dilute! | * Aliquot 375 μL of "Thermo pol buffer" into tubes—do '''not''' dilute! | ||
Note: Store in the | Note: Store in the –20°C freezer in the box marked "Other Enzymes" | ||
===10X ''Pfx'' buffer=== | ===10X ''Pfx'' buffer=== | ||
* Label tubes: 10X ''Pfx'' buffer | * Label tubes: 10X ''Pfx'' buffer | ||
* Thaw and mix (stir) "''Pfx50'' 10X PCR Buffer" (NEB; in freezer) | * Thaw and mix (stir) "''Pfx50'' 10X PCR Buffer" (NEB; in freezer) | ||
* Aliquot 375 μL of "''Pfx50'' 10X PCR Buffer" into tubes—do '''not''' dilute! | * Aliquot 375 μL of "''Pfx50'' 10X PCR Buffer" into tubes—do '''not''' dilute! | ||
Note: Store in the | Note: Store in the –20°C freezer in the box marked "Other Enzymes" | ||
===8X DNA loading=== | ===8X DNA loading=== | ||
* 40% [[Griffitts:Stock solutions#80% Glycerol (75 mL)|glycerol]] | * 40% [[Griffitts:Stock solutions#80% Glycerol (75 mL)|glycerol]] | ||
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* 10 mL N,N-dimethylformamide (DMF)(in fume hood) | * 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) | * 0.2 g 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-gal)(in freezer) | ||
Store at | Store at –20°C | ||
===0.2 M IPTG=== | ===0.2 M IPTG=== | ||
* 5 mL ddH<sub>2</sub>O | * 5 mL ddH<sub>2</sub>O | ||
* 0.238g isopropyl-β-D-thiogalactopyranoside (IPTG)(in freezer) | * 0.238g isopropyl-β-D-thiogalactopyranoside (IPTG)(in freezer) | ||
Sterile filter, aliquot, and store at | Sterile filter, aliquot, and store at –20°C | ||
===1 M Copper (II) sulfate (50 mL)=== | ===1 M Copper (II) sulfate (50 mL)=== | ||
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* 771 mg Dithiothreitol (DTT; in fridge) | * 771 mg Dithiothreitol (DTT; in fridge) | ||
Aliquot 500 μL into Eppendorf tubes | Aliquot 500 μL into Eppendorf tubes | ||
Store in | Store in –20°C | ||
===Orcinol spray=== | ===Orcinol spray=== |
Revision as of 09:32, 17 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)
- 1.0 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 | 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 | 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 | 5 mg/mL | 30 μg/mL (1000X) | N/A | ddH2O | 4°C | |
Neomycin | Nm | GREEN | 25 mg/mL | N/A | 100 μg/mL (250X) | ddH2O | 4°C |
Rifampicin | Rm | 50 mg/mL | N/A | 100 μg/mL (500X) | 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 | 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. Exceptions: Gentamycin, Nalidixic Acid, and Rifampicin.
DNA
10 mM dNTPs
- Thaw and mix (stir) 100 mM stock solutions of dGTP, dATP, dTTP, dCTP (NEB; in freezer)
- Master Mix:
- Dispense 100-μL aliquots into 1.7-mL tubes
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