• General Buffers and Stock Solutions
  • DNA extraction buffers
  • Sugar stock solutions
  • 50X TAE
  • Gels
• Antibiotic Stocks
• E.coli Media
  • Luria Broth Plates
  • Luria Broth
  • 5X Supplement EZ (1000 ml)
  • Top Agar
  • Minimal Defined Media for E.coli
• S.cerevisiae Media
  • YPD liquid media(for 400mL)
  • YPD Agar (for 400ml)
  • 10x YNB(for 400ml) (w/ ammonium sulfate)
  • SC Agar (Synthetic Complete 400ml)
  • SD Agar(Synthethic Defined 400mL)
• C.reinhartii Media

General Buffers and Stock Solutions

DNA extraction buffers

1. 50 mM Tris HCl and 10 mM EDTA all in 250 ml.

• 157.6 grams (molecular weight of Tris HCl) x (50mM) x (1/1000mMol) x 1/4 = 1.97 grams
• (292.25g) x (10 mM) x (1/1000) x (1/4) = .73 grams

Note: PH must be adjusted for Edta to dissolve.

2. 200mM NaOH + 1% w/v SDS
* 40g * 200mmol * 1/1000 * 1/4 = 2 grams NaOH
* 250 ml * .01 = 2.5 g SDS

3. 3M KAc
* 3M * 98.14 * 1/4 = 73.605
* 30ml of glacial acetic acid to make PH go down to 5.5

4. "Faux" SOC Media
* 9mL LB
* 1mL 40% glucose stock solution
* 100uL 1M MgCl₂
* 100uL 1M MgSO₄

Sugar stock solutions

• 40% Dextrose Solution
  • 60g Dextrose into 150 mL of H₂O
  • Filter Sterilize through the Nalgene 50mm Filter Unit
  • Refrigerate

50X TAE

• 242 g Tris base
• 57.1 mL glacial acetic acid
• 37.2 g Na₂EDTA.2H₂0
• H₂0 to 1 Liter mark

Gels

• 1g LE Agarose
• 100 mL of 1x TAE
• Microwave:
  • Always use a flask to prepare the gel.
  • Remove Aluminum Foil Top
  • Unscrew, but leave bottle cap on
  • Microwave for < 1 min (first boil)
  • Swirl contents of bottle
  • Microwave again for 15 seconds (second boil)
  • Let Cool
• add 5µL ethidium bromide
  • Swirl contents of bottle

Antibiotic Stocks

All community stocks can be found in Freezer A

• Ampicillin (100mg/mL)
• Spectinomycin (100 mg/mL)
• Chloramphenicol (30 mg/mL in Ethanol)
• Kanamycin (50mg/mL)

E.coli Media

Made in 400 ml solution

Luria Broth Plates

• 4g Bacto Tryptone
• 4g Sodium chloride
• 2g Yeast Extract
• 6g Bacto Agar
• 400mL water

Autoclave

Luria Broth

• 4g Bacto Tryptone
• 4g Sodium chloride
• 2g Yeast Extract
• 400mL water

Autoclave

5X Supplement EZ (1000 ml)

Stock Solution	Volume (mL)
r2c1	r2c2

Stock Solution
Alanine
Arginine
Asparagine
Aspartic Acid
Cysteine
Glutamic Acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
Water
Total

Top Agar

• 1g Bacto Tryptone
• 0.5g Yeast Extract
• 1g Sodium Chloride
• 0.7g Bacto Agar
• Dissolve in 100mL distilled water

Autoclave


!!

• 100x dilution of SOB with 2M glucose added.

Minimal Defined Media for E.coli

• 500 mL Total
  • 5 mL of 40% Dextrose Solution
  • 100 mL of 5x M9 Salt Solution
  • 1 mL of MgSO₄
  • 500 µL of CaCl₂
  • Add Water to 500 mL
  • 2.5 microliter thiamine

S.cerevisiae Media

YPD liquid media(for 400mL)

• 4g Bacto Yeast Extract
• 8g Bacto Peptone
• 400mL H20
• Autoclaved to sterilize
• 8g filter sterilized glucose (20mL 40% soln)

YPD Agar (for 400ml)

• 4g Bacto Yeast Extract
• 8g Bacto Peptone
• 8g Bacto Agar
• Autoclave to sterilize
• 8g Dextrose

10x YNB(for 400ml) (w/ ammonium sulfate)

• 26.8 g base
• 400 ml H₂0
• AUTO-CLAVE - remember to shake and loosen cap before starting machine

SC Agar (Synthetic Complete 400ml)

• 8g Bacto Agar
• 300 ml H20 (NOTE- should be adding less to account for a total 400ml soln.)
• _AUTO-CLAVE_

Place on a stir plate. Add sterile stir bar and mix slowly (too fast makes bubbles in the agar!). Allow to cool to ~55^oC - Then with a sterile pipet, add

• 40mL 10x YNB (w/ ammonium sulfate)
• 8g dextrose (20mL 40% dextrose solution)
• 20 Amino acids

SD Agar(Synthethic Defined 400mL)

• 8g Bacto Agar
• 300 ml H20 (NOTE- should be adding less to account for a total 400ml soln.)
• _AUTO-CLAVE_

Place on a stir plate. Add sterile stir bar and mix slowly (too fast makes bubbles in the agar!). Allow to cool to ~55^oC - Then with a sterile pipet, add

• 40mL 10x YNB (w/ ammonium sulfate)
• 8g dextrose (20mL 40% dextrose solution)
• Whatever combination of Supplements
  • 1mL Lysine stock
  • 8mL Leucine Stock
  • 4mL of the .2%(W/V) Uracil Stock

C.reinhartii Media