Griffitts:Stock solutions

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Antibiotics

Ampicillin (40 mL)

For a stock solution of 25 mg/mL:

NOTE: Store at 4°C

Chloramphenicol (40 mL)

For a stock solution of 7.5 mg/mL:

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:

NOTE: Store at 4°C

Hygromycin (40 mL)

For a stock solution of 5 mg/mL:

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:

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:

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:

NOTE: Store at 4°C

Streptomycin (40 mL)

For a stock solution of 50 mg/mL:

NOTE: Store at 4°C

Tetracycline (40 mL)

For a stock solution of 2.5 mg/mL:

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:

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)
or
25 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:
  • 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

DNA ladder

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
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