Stock Solution
Antibiotics
Ampicillin (40 mL)
For a stock solution of 25 mg/mL:
- 1 g Ampicillin
- 40 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Chloramphenicol (40 mL)
For a stock solution of 7.5 mg/mL:
- 300 mg Chloramphenicol
- 40 mL 50% Ethanol
NOTE: Do not filter sterilize—the ethanol may dissolve the filter
NOTE: Store at 4°C
Erythromycin (20 mL)
For a stock solution of 10 mg/mL:
- 200 mg Erythromycin
- 20 mL 100% ethanol (in fume hood)
- Do not filter sterilize—the ethanol may dissolve the filter
NOTE: Store at –20°C
Gentamicin (20 mL)
For a stock solution of 7.5 mg/mL:
- 150 mg Gentamicin
- 20 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Hygromycin (40 mL)
For a stock solution of 5 mg/mL:
- 200 mg Hygromycin
- 40 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Kanamycin (40 mL)
For a stock solution of 7.5 mg/mL:
- 300 mg Kanamycin
- 40 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Nalidixic acid (30 mL)
For a stock solution of 30 mg/mL:
- 900 mg Nalidixic Acid
- 360 mg NaOH
- 30 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Neomycin (40 mL)
For a stock solution of 25 mg/mL:
- 1 g Neomycin
- 40 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Phosphomycin
For a stock solution of 25 mg/mL:
- 1 g Phosphomycin
- 40 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Rifampicin (20 mL)
For a stock solution of 50 mg/mL:
- 1 g Rifampicin
- 20 mL N,N-dimethylformamide (DMF; in fume hood)
NOTE: Do not filter sterilize—the DMF will dissolve the filter
NOTE: Store at –20°C
Spectinomycin (40 mL)
For a stock solution of 20 mg/mL:
- 800 mg Spectinomycin
- 40 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Streptomycin (40 mL)
For a stock solution of 50 mg/mL:
- 2 g Streptomycin
- 40 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Tetracycline (40 mL)
For a stock solution of 2.5 mg/mL:
- 100 mg Tetracycline
- 40 mL 50% Ethanol
NOTE: Do not filter sterilize—the ethanol may dissolve the filter
NOTE: Store at 4°C
Triclosan (40 mL)
For a stock solution of 10 mg/mL:
NOTE: Do not filter sterilize—the ethanol may dissolve the filter
NOTE: Store at 4°C
Trimethoprim (40 mL)
For a stock solution of 6.25 mg/mL:
- 250 mg Trimethoprim
- 40 mL 100% DMSO
NOTE: Do not filter sterilize—the DMSO may dissolve the filter
NOTE: Store at 4°C
Antibiotics Table
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 "PCR Supplies"
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 "PCR Supplies"
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 "PCR Supplies"
5X
Q5
buffer
- Label tubes: 5X Q5 buffer
- Thaw and mix (stir) "Q5 5X PCR Buffer" (NEB; in freezer)
- Aliquot 100 μL of "Q5 5X PCR Buffer" into tubes—do not dilute!
Note: Store in the –20°C freezer in the box marked "PCR Supplies"
8X DNA loading
- 40% glycerol
- 1/50 dilution of 2% bromophenol blue
DNA ladder
- 140 μL ddH20
- 40 μL 8X DNA loading buffer
- 20 μL 1 kb ladder from NEB
- 2 μL 0.25 M EDTA
Note: use 4 μL per lane; 3kb band = 50 ng
Ethidium Bromide (1 mL)
- 100 μL 10 mg/mL ethidium bromide
- This is stored on the middle shelf of the refrigerator on the right
- 900 μL ddH2O
Note: use 8 μL per 50 mL gel
WARNING: Always wear gloves when handling ethidium bromide
Ethidium Bromide (10 mL)
- 10 mL ddH2O
- 10 mg ethidium bromide
Note: use 8 μL per 50 mL gel
WARNING: Always wear gloves when handling ethidium bromide
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)
- 35 mL ddH2O
- 11.76 g sodium citrate tribasic dihydrate (Na3C6H5O7·2H2O)
- pH to 5.7 with HCl (~1 mL)
- QS to 40 mL with ddH2O
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%
(75 mL)
- 75 mL ddH2O
- 7.5 g Sodium dodecyl sulfate (SDS)
Do not sterilize
20%
(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