SC-Leu low-Ade plates

From Jeff Laney's recipe:

(BUT, can't make 10X SC-AHLT)

Per liter of medium, mix the following asceptically, and pre heat:

Component	Volume (mL)
10X SC-AHLT	100
10X YNB+AS	100
10X 20% glucose	100
10X Adenine hemi sulfate	2.5
50X Tryptophan	20
100X Histidine HCl	10

Add to 20g agar in 667.5 mL water, autoclaved 30 minutes

Mark with one green and one blue line.

SC -Leu low Ade 4x + powder

Use the 4x SC-AHLT to mix dry ingredients

(4x already made; not enough dry left to start over.)

per Liter:

Agar 20 g + 617.5 mL water. Autoclave

Meanwhile, make:

Component	Volume (mL)
4X SC-AHLT	250
10X Adenine hemi sulfate	2.5
10X 20% glucose	100
50X Tryptophan	20
100X Histidine HCl	10

Add dry:

Component	g
YNB	1.7
Ammonium Sulfate	5

Filter sterilize, warm up, then add to molten agar

Mark with one green and one blue line

Using 4X SC-AHLT because 10x won't go into solution:

(BUT: Agar is too viscous - bubbles won't rise to the surface before it sets up.)

To make	SC-leu low ade agar plates
with	mm plate diameter
pour	mL per plate
start with:	mL water
add:	g agar, leave stir bar in
autoclave for:	minutes
Meanwhile, using	X SC-AHLT
Asceptically mix together, warm up, then add to autoclaved agar:	mL SC-AHLT,
and :	mL 10X YNB+AS,
and:	mL 20% Glucose,
and:	mL 10x adenine hemi sulfate,
and:	mL 50X Tryptophan
and:	mL 100X Histidine .
for a final volume of	mL

Mark with one green and one blue line.

High concentration agar is difficult to work with.

• Tends to boil over, so needs a lot of water in the autoclave pan
• Hard to get into solution - frequently needs re-autoclaving
• So viscous that bubbles don't rise to the surface

So

• mix the ingredients that can't be autoclaved in as little water as possible
• Only use high concentration stock solutions for micro-ingredients

Per Liter:

dry ingredient	g
SC-AHLT	1.64
YNB	1.7
Ammonium sulfate	5
glucose	20

stock solutions	mL
10X Adenine hemi sulfate	2.5
50X Tryptophan	20
100X Histidine HCl	10

plus water to 150 mL

filter sterilize

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Autoclave

• 20 g agar
• 850 mL water

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Mix and pour

Mark with one green and one blue line.