Team:Paris Bettencourt/Notebook/Drug Screening/Wednesday 24th July.html

Drug Screening

ASDF

Day Number^suffix Month

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24/7/13

Made 500 ml of M9 Minimal Plates with Sulfur Dropout Powder accouding to the following recipe:

Recipe: M9 Minimal Plates with Sulfur Dropout Powder

These stock solutions can be pre-sterilized and stored indefinitely:

5x M9 Salts (from scratch)

Reagent	Quantity
Na2HPO4	17 g
KH2PO4	7.5 g
NaCl	1.25 g
(NH4)2SO4	3.0 g

Bring to a total volume of 500 ml.

Adjust pH to 7.4 with NaOH.

Sterilize by autoclaving or filtration

or

5x M9 Salts (from Difco Powder)

Reagent	Quantity
M9 Salts	28.2 g

Bring to a total volume of 500 mL.

Sterilize by autoclaving or filtration

2x Yeast Plate Agar

Reagent	Quantity
Agar	30 g

Bring to a total volume of 500 ml.

Sterilize by autoclaving

5x Amino Acid Dropout Powder

Reagent	Quantity
Sulfur dropout powder	0.5 g

Bring to a total volume of 200 ml.

Sterilize by autoclaving

Sulfur dropout powder is a rich supplement mix with cysteine and methionine omitted:

Amino acids		Nucleotide bases
Alanine	2.0 g	Leucine	10.0 g	Adenine	0.5 g
Arginine	2.0 g	Lysine	2.0 g	Uracil	2.0 g
Asparagine	2.0 g	Methionine	0.0 g
Aspartic acid	2.0 g	Phenylalanine	2.0 g	Vitamins
Cysteine	0.0 g	Proline	2.0 g	p-Aminobenzoic acid	0.2 g
Glutamic acid	2.0 g	Serine	2.0 g	Inositol	2.0 g
Histidine	2.0 g	Tyrosine	2.0 g
Isoleucine	2.0 g	Valine	2.0 g

Recipe: M9 Minimal Plates with Sulfur Dropout Powder

The plates can be made fresh from pre-sterilized stock solutions.

Reagent	Quantity
5x M9 Salts	200 mi
5x Amino Acid Dropout Powder	200 mi
1 M MgSO4	2 ml
20% Glucose	20 ml
1M CaCl2	0.1 ml

Bring to a total volume of 500 mL

Stir and warm on a hot plate to about 60 C (hot to the touch)

Melt 500 ml 2x Yeast Plate Agar in the microwave for ~8 minutes on high

Cool to about 60 C (hot to the touch)

This takes about 30 minutes on the bench or 5 minutes in a water bath.

Add the 2x Yeast Plate agar to the other reagents to make 1 L of media.

This recipe will prepare 20 plates at 25 ml / plate.

made 7 plates without antibiotics and 20 with Chloramphenicol and Spectinomycin - by mistake.

Used one plate without AB to plate the following strains:

• sD007=sD001+pD005

• sD008=sD001+pD006

• sD013= sD001+pD005+ pD004

• sD015= sD001+pD006+ pD004