Griffitts:Stock solutions

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:

• 400 mg Triclosan
• 40 mL 100% Ethanol

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

Antibiotic Specifications

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	50% EtOH	4°C
Erythromycin	Er	(none)	10 mg/mL	N/A	N/A	100% 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)	Use Neomycin	ddH2O	4°C
Nalidixic acid	Na	(none)	30 mg/mL	30 μg/mL (1000X)	N/A	ddH2O	4°C
Neomycin	Nm	GREEN	25 mg/mL	Use Kanamycin	100 μg/mL (250X)	ddH2O	4°C
Phosphomycin	Pm	(none)	25 mg/mL	N/A	100 μg/mL (250X)	ddH2O	4°C
Rifampicin	Rf	(none)	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	5 μg/mL (500X)	5 μg/mL (500X)	50% EtOH	4°C
Triclosan	Tr	(none)	10 mg/mL	N/A	N/A	100% EtOH	4°C
Trimethoprim	Tm	(none)	6.25 mg/mL	N/A	12.5 μg/mL (500X) or25 μg/mL (250X)	100% DMSO	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:
  • 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

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% 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