Recipes for reagents formerly made by the student helper

Note: This page is kind of a mess. If you need information on the reagents listed here, please follow the links in their names.

YEP Liquid Media (1L)

20 g Bacto Peptone (powder)

10 g Bacto Yeast Extract (powder)

1) Put a little (~100 ml) diH₂O in 1L beaker

2) Add powders and stir bar.

3) As it stirs, add diH₂O to 960 mL mark (to minimize lumps)

4) When completely clear, pour into bottles (note: only 400 ml can go in a 500 ml bottle)

5) Autoclave 45 minutes (probably less is okay)

Sporulation Media (1L) 3 g Potassium Acetate (KOAc or CH₃COOK)

0.2 g Raffinose

1) Mix 500 ml diH₂O, KOAc, and raffinose in 1000 ml beaker, dissolve

2) Bring volume to one liter with more diH₂O

3) Filter sterilize into 4 250 ml bottles

0.5M EDTA (1L) 186.1 g Na₂EDTA·2H₂O stirring in ~800 ml milliQ-pure H₂O in a 1 liter beaker. Add 20 g NaOH pellets, which will allow dissolution of the remainder of the EDTA. Now add additional NaOH, in the form of 50% solution to bring the pH to 8.0 and bring to volume. Autoclave in two 500 ml bottles

10X TE (1L)

10 ml 1M Tris-HCl, pH = 8.00

5 ml 0.2M EDTA (2 ml of 0.5M EDTA)

~1000 ml diH₂O Milli-Q

1) Combine Tris-HCl, EDTA, and Milli-Q water in one liter beaker

2) Autoclave or filter sterilize

20% SDS

For 4260 ml:

Sigma L-5750

diH₂O

2L beaker lined w/ plastic bag weigh 852 g SDS. Fill 4L jug part way. Add stir bar. Using funnel, add 850 g SDS. Stir O/N. Continue adding until dissolved. Bring to volume. Aliquot to 1L bottles. No autoclave.

Smash ‘n’ Grab Lysis Buffer (80 ml)

0.8 ml 1M Tris pH 8 ( -> 10 mM)

160 µl 0.5M EDTA (-> 1 mM)

1.6 ml 5M NaCl (-> 0.1M)

8 ml Triton X100 20% (->2%)

4 ml 20% SDS (-> 1%)

65.2 ml diH₂O

No need to be autoclaved. Place 2 x 40 ml into Falcon tubes

1M LiOAc 20.4 g LiAc

10.2 g H₂O to 200 ml Milli Q H₂O to 100 ml

Autoclave (I, too am confused be this recipe as I am transcribing it – Brett)

50% PEG 1) Place 50 g polyethylene glycol powder in beaker

2) Add 30-40 ml diH₂O

3) Heat and stir until totally clear. (It takes forever)

4) Dilute to 100 ml with diH₂O (wait about 40 min & till gets clear)

5) Transfer to a 125 ml bottle using a steriflip vacuum filter

Note: polyethylene glycol comes in a big tub

50% Glucose (400 ml, in 2 x 200 ml) 1) 200 g Dextrose into 200 ml heated diH_2­O (heat in microwave for 1 min)

2) Stir for awhile

3) Bring to 400 ml

4) Stir until clear

5) Filter sterilize into bottles

80% Glycerol

Glycerol from stock shelf

1) Microwave entire bottle 1 min

2) 8 parts glycerol to 2 parts diH₂O

3) pipet using glass pipettes for volume ~100 ml. Autoclave 20 minutes

Luria Broth (LB bacteria media)

10 g Bacto Tryptone (powder)

5 g Bacto Yeast Extract (powder)

5 g NaCl (crystals)

160 µl 50% NaOH solution (very basic- wear gloves)

1) Dissolve into 1 L tap diH₂O

2) Autoclave 35 minutes

Tris-buffer, Acetic Acid, EDTA (50X TAE 1 liter)

242 g Tris Base (powder)

57 ml HOAc – glacial acetic acid solution (under hood in cabinet)

100 ml 0.5M EDTA (solution)

Water to 1 L

This makes 50X TAE of pH 8.4. Do not autoclave or filter sterilize