Griffitts:Stock solutions

Ampicillin (40 mL)
For a stock solution of 25 mg/mL: NOTE: Store at 4°C
 * 1 g Ampicillin
 * 40 mL ddH2O
 * Filter sterilize

Chloramphenicol (40 mL)
For a stock solution of 7.5 mg/mL: NOTE: Do not filter sterilize&mdash;the ethanol may dissolve the filter NOTE: Store at 4°C
 * 300 mg Chloramphenicol
 * 40 mL 50% Ethanol

Erythromycin (20 mL)
For a stock solution of 10 mg/mL: NOTE: Store at –20°C
 * 200 mg Erythromycin
 * 20 mL 100% ethanol (in fume hood)
 * Do not filter sterilize&mdash;the ethanol may dissolve the filter

Gentamicin (40 mL)
For a stock solution of 7.5 mg/mL: NOTE: Store at 4°C
 * 300 mg Gentamicin
 * 40 mL ddH2O
 * Filter sterilize

Hygromycin (40 mL)
For a stock solution of 5 mg/mL: NOTE: Store at 4°C
 * 200 mg Hygromycin
 * 40 mL ddH2O
 * Filter sterilize

Kanamycin (40 mL)
For a stock solution of 7.5 mg/mL: NOTE: Store at 4°C
 * 300 mg Kanamycin
 * 40 mL ddH2O
 * Filter sterilize

Nalidixic acid (30 mL)
For a stock solution of 30 mg/mL: NOTE: Store at 4°C
 * 900 mg Nalidixic Acid
 * 360 mg NaOH
 * 30 mL ddH2O
 * Filter sterilize

Neomycin (40 mL)
For a stock solution of 25 mg/mL: NOTE: Store at 4°C
 * 1 g Neomycin
 * 40 mL ddH2O
 * Filter sterilize

Phosphomycin
For a stock solution of 25 mg/mL: NOTE: Store at 4°C
 * 1 g Phosphomycin
 * 40 mL ddH2O
 * Filter sterilize

Rifampicin (20 mL)
For a stock solution of 50 mg/mL: NOTE: Do not filter sterilize&mdash;the DMF will dissolve the filter NOTE: Store at –20°C
 * 1 g Rifampicin
 * 20 mL N,N-dimethylformamide (DMF; in fume hood)

Spectinomycin (40 mL)
For a stock solution of 20 mg/mL: NOTE: Store at 4°C
 * 800 mg Spectinomycin
 * 40 mL ddH2O
 * Filter sterilize

Streptomycin (40 mL)
For a stock solution of 50 mg/mL: NOTE: Store at 4°C
 * 2 g Streptomycin
 * 40 mL ddH2O
 * Filter sterilize

Tetracycline (40 mL)
For a stock solution of 2.5 mg/mL: NOTE: Do not filter sterilize&mdash;the ethanol may dissolve the filter NOTE: Store at 4°C
 * 100 mg Tetracycline
 * 40 mL 50% Ethanol

Triclosan (40 mL)
For a stock solution of 10 mg/mL: NOTE: Do not filter sterilize&mdash;the ethanol may dissolve the filter NOTE: Store at 4°C
 * 400 mg Tetracycline
 * 40 mL 100% Ethanol

Trimethoprim (40 mL)
For a stock solution of 6.25 mg/mL: NOTE: Do not filter sterilize&mdash;the DMSO may dissolve the filter NOTE: Store at 4°C
 * 250 mg Trimethoprim
 * 40 mL 100% DMSO

Antibiotics Table
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.

10 mM dNTPs
Note: Store in the –20°C freezer in the box marked "Other Enzymes"
 * Thaw and mix (stir) 100 mM stock solutions of dGTP, dATP, dTTP, dCTP (NEB; in freezer)
 * Master Mix:
 * 60 μl sterile dH2O
 * 10 μl dGTP
 * 10 μl dATP
 * 10 μl dTTP
 * 10 μl dCTP
 * Dispense 100-μL aliquots into 1.7-mL tubes

10X Taq buffer
Note: Store in the –20°C freezer in the box marked "Other Enzymes"
 * Label tubes: 10X Taq buffer
 * Thaw and mix (stir) "Thermo pol buffer" (NEB; in freezer)
 * Aliquot 375 μL of "Thermo pol buffer" into tubes&mdash;do not dilute!

10X Pfx buffer
Note: Store in the –20°C freezer in the box marked "Other Enzymes"
 * 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&mdash;do not dilute!

8X DNA loading

 * 40% glycerol
 * 1/50 dilution of 2% bromophenol blue

DNA ladder
Note: use 5 μL per lane; 3kb band = 60 ng
 * 140 μL ddH20
 * 40 μL 8X DNA loading buffer
 * 20 μL 1 kb ladder from NEB
 * 2 μL 0.25 M EDTA

Ethidium Bromide (1 mL)
Note: use 8 μL per 50 mL gel WARNING: Always wear gloves when handling ethidium bromide
 * 100 μL 10 mg/mL ethidium bromide
 * This is stored on the middle shelf of the refrigerator on the right
 * 900 μL ddH2O

Ethidium Bromide (10 mL)
Note: use 8 μL per 50 mL gel WARNING: Always wear gloves when handling ethidium bromide
 * 10 mL ddH2O
 * 10 mg ethidium bromide

1 M Calcium Chloride (75 mL)
Autoclave
 * 75 mL ddH2O
 * 11.027 g calcium chloride dihydrate (CaCl2·2H2O)

2 M Magnesium Chloride (75 mL)
Autoclave
 * 75 mL ddH2O
 * 30.4965 g magnesium chloride hexahydrate (MgCl2·6H2O)

1 M Sodium Citrate (40 mL)
Sterile filter
 * 40 mL ddH2O
 * 11.76 g sodium citrate tribasic dihydrate (Na3C6H5O7·2H2O)
 * pH to 5.7 with HCl

2% X-gal
Store at –20°C
 * 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 M IPTG
Sterile filter, aliquot, and store at –20°C
 * 5 mL ddH2O
 * 0.238g isopropyl-β-D-thiogalactopyranoside (IPTG)(in freezer)

1 M Copper (II) sulfate (50 mL)
Sterile filter
 * 50 mL ddH2O
 * 7.98 g CuSO4

10% SDS (75 mL)
Do not sterilize
 * 75 mL ddH2O
 * 7.5 g Sodium dodecyl sulfate (SDS)

20% SDS (75 mL)
Do not sterilize
 * 75 mL ddH2O
 * 15 g Sodium dodecyl sulfate (SDS)

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)
Autoclave
 * 75 mL ddH2O
 * 21.92 g NaCl

80% Glycerol (75 mL)
Microwave for 30-second intervals on high until dissolved Autoclave
 * 15 mL ddH2O
 * 60 mL glycerol

1 M DTT (5 mL)
Aliquot 500 μL into Eppendorf tubes Store in –20°C
 * 5 mL ddH2O
 * 771 mg Dithiothreitol (DTT; in fridge)

Orcinol spray

 * 5 mL ddH2O
 * 25 mL methanol (in fume hood)
 * 10 mL sulfuric acid (H2SO4 (in fume hood&mdash;be careful!)
 * 200 mg orcinol