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

From 2013.igem.org

Drug Screening

Wednesday 24th July

Place your twit here

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