Reagents

Vitamin Solution (PES30)

Biotin 10 mg
Thiamine 200 mg
Cyanocobalamin 10 mg
Folic Acid 10 mg
Myo-Inositol 10 mg
Calcium Pantothenate 100 mg
Milli-Q Water ~1 L
  • Measure out 500 mL Milli-Q
  • Add components in order
  • Bring volume up to 1 L with Milli-Q
  • Filter sterilize before use

Vitamin Solution (IMR)

Thiamine•HCl (20 g/L) 1 mL
Biotin (50 mg/L) 2 mL
Vitamin B12 (1 g/L) 100 µL
Milli-Q Water 96.9 mL
  • Combine all ingredients
  • Filter sterilize the solution
  • Store at 4oC

1M Tris

Tris Base 121.1 g
Milli-Q Water ~1 L
HCl ~42 mL
  •  Dissolve Tris base into 800 mL Milli-Q water
  • Using HCl, adjust pH to 8.0
  • Bring volume to 1 L using Milli-Q
  • Autoclave for 30 minutes (NOTE: re-make if solution turns yellow)
  • Store at 4oC

Trace Metal Solution (IMR)

CuSO4•5H2O (9.80 g/L) 1 mL
ZnSO4•7H2O (22 g/L) 1 mL
CoCl2•6H2O (10 g/L) 1 mL
MnCl2•7H2O (244 g/L) 1 mL
Na2MoO4•7H2O (6.3 g/L) 1 mL
Fe2(SO4)3•nH2O (900 mg/L) 500 mL
EDTA•Na2 (6.7 g/L) 500 mL
  • Combine last two ingredients
  • Add all other ingredients in order
  • Store at 4oC

Trace Metal Solution A5+Co

H3BO3 2.86 g
MnCl2•4H2O 1.81 g
ZnSO4•7H2O 222 mg
Na2MoO4•2H2O 390 mg
CuSO4•5H20 79 mg
Co(NO3)2•6H2O 49 mg
Milli-Q Water ~1 L

 

  •  Measure out 500 mL Milli-Q
  • Add components in order
  • Bring volume to 1 L with Milli-Q

TESC

NaCl 87.66 g
1M Tris 100 mL
0.5M EDTA 200 mL
Hexadecyltrimethylammonium Bromide (CTAB) 10 g
Milli-Q Water ~1 L
  • Measure out approximately 500 mL Milli-Q
  • Add all components in order
  • Bring volume up to 1 L with Milli-Q
  • Adjust pH to ~8.0
  • Autoclave for 30 minutes
  • Store at room temperature

TE Buffer

1M Tris 5 mL
0.5M EDTA 1 mL
Milli-Q Water ~500 mL

 

  • Measure out 490 mL Milli-Q
  • Add components in order
  • Adjust pH to 7.4 using HCl
  • Bring volume to 500 mL
  • Autoclave for 30 minutes
  • Store at 4oC

TBE

Trizma Base 12.11 g
Boric Acid, anhydrous 5.565 g
EDTA 372 mg
Milli-Q Water ~1 L

 

  • Measure out approximately 300 mL Milli-Q
  • Add all components in order
  • Bring volume up to 1 L with Milli-Q
  • Adjust pH to 8.3
  • Store at 4o

TAE

Trizma Base 4.844 g
EDTA 372.2 mg
Glacial Acetic Acid 1.15 mL
Milli-Q Water ~1 L
  • Measure out approximately 300 mL Milli-Q
  • Add all components in order
  • Bring volume up to 1 L with Milli-Q
  • Adjust pH to 8.3
  • Store at 4oC

10% SDS

Sodium Dodecyl Sulfate (SDS) 25 g
Milli-Q Water ~250 mL
  • Pour 175 mL Milli-Q into sterile 250 mL glass bottle
  • Put on mask (very important) and weigh out SDS
  • Thoroughly clean countertop and balance
  • Add SDS to the Milli-Q and heat to dissolve
  • Bring volume to 250 mL with Milli-Q
  • Adjust pH to 7.2 using sterile HCL (1% v/v) and/or NaOH (1% w/v)
  • Loosen cap and microwave for one to two minutes
  • Store at room temperature

25% Ringer Solution

NaCl 2.25 g
KCl 105 mg
CaCl2 45 mg
NaHCO3 50 mg
Citric Acid 34 mg
Milli-Q Water ~1 L
  • Measure out approximately 300 mL Milli-Q
  • Add all components in order
  • Bring volume up to 1 L with Milli-Q
  • Autoclave for 45 minutes

-Pi Stock

MgSO4•7H2O 20 g
CaCl2•2H2O 6 g
NaCl 20 g
Microelement Stock 500 mL
Milli-Q Water 1.5 L

 

  •  Dilute first three ingredients separately into 500 mL of Milli-Q
  • Autoclave three solutions, and allow to cool
  • Combine four solutions in order, allowing each to dissolve
  • Store at 4oC, shake well before use, do not autoclave

+Pi Stock

2HPO4•3H2O 28 g
K2HPO4, anhydrous 21.4 g
Milli-Q Water 500 mL
  • Measure out Milli-Q
  • Add either of two chemicals
  • Autoclave both containers
  • Store at 4oC

PES Stock Solution

NaNO3 50 mg
Na2-Glycerolphosphate•4H2O 50 mg
Tris Buffer 500 mg
Iron Solution 25 mL
Trace Metal 15 mL
Milli-Q Water 100 mL
  • Measure out Milli-Q
  • Add NaNO3 and Na2-Glycerolphosphate•4H2O
  • Combine trace metal and iron solutions in separate container
  • Autoclave both containers
  • Once cool, combine both mixtures
  • Store at 4oC in 10 mL aliquots

PCI Solution (25:24:1)

Isoamyl Alcohol 5 mL
Chloroform 120 mL
Phenol 125 mL

 

  • Put on gloves, cover forearms, and relocate to fume hood
  • Combine all components in sterile 250 mL glass bottle
  • Mix well
  • Wrap container in foil to protect from light
  • Store at 4oC

Micronutrient Solution

H2SO4 500 µL
H3BO3 500 mg
MnSO4•H2O 2.28 g
ZnSO4•7H2O 500 mg
CuSO4•5H2O 25 mg
Na2MoO4•2H2O 25 mg
CoCl2•6H2O 45 mg
Na2SeO4 4 mg
Milli-Q Water ~1 L
  • Measure out 500 mL of Milli-Q
  • Dissolve all components in order
  • Bring volume to 1 L with Milli-Q

Microelement Stock

Fe-EDTA solution 80 mL
MnCl2•4H2O 180 mg
MoO3 (85% purity) 18 mg
or
Na2Mo4•2H2O (99% purity)
30.55 mg
ZnSO4•7H2O 22.0 mg
CuSO4•5H2O 7.9 mg
H3BO3 (Boric Acid) 286 mg
Vanadyl sufate hydrate 6.6 mg
CoC12•6H2O 4.0 mg
Milli-Q Water ~545 mL
  • Measure out Milli-Q
  • Dissolve all components in order
    (Note: Do Not autoclave)

Iron Solution

FeNH4(SO4)2•10H2O 351 mg
Na2EDTA 300 mg
Milli-Q Water ~500 mL
  • Measure out 250 mL Milli-Q
  • Add components in order
  • Bring volume up to 500 mL
  • Autoclave for 25 minutes

Fe-EDTA (A&A)

Na2EDTA•2H2O 866.7 mg
FeSO4•7H2O
or
Fe2(SO4)3•nH2O
2.28 mg
KOH 866.7 mg
Milli-Q Water ~100 mL
  • Dissolve KOH in 31 mL Milli-Q
  • Add Na2EDTA•2H2O
  • In separate container, dissolve FeSO4•7H2O or Fe2(SO4)3•nH2O in 60.67 mL Milli-Q
  • Mix both solutions together
  • Bubble filtered air through combined solutions until the color darkens (~2.5 hr)

0.5M EDTA

Na2EDTA- dihydrate 186.1 g
Milli-Q Water ~1 L
NaOH ~20 g
  • Add EDTA into 800 mL Milli-Q water (NOTE: EDTA will not dissolve)
  • Using NaOH pellets, adjust pH to 8.0 (EDTA should now dissolve)
  • Bring volume to 1 L with Milli-Q
  • Autoclave for 30 minutes
  • Store at 4oC

CI Solution (24:1)

Isoamyl Alcohol 10 mL
Chloroform 240 mL
  • Put on gloves, cover forearms, and relocate to fume hood
  • Add both components to a sterile 250 mL glass bottle
  • Mix well
  • Wrap container in foil to protect from light
  • Store at 4oC