Protocol
DC title: protocol for the preparation of Saccharomyces cerevisiae competent cells
DC author: Wayne Aubrey
DC organisation: Aberystwth University
status: draft
DC submission date: 15 January 2008


Order of the operating procedures
make YPD agar plates
make single colonies plates
grow yeast culture
calibrate spectrophotometer
measure yeast culture optical density
make yeast culture
calibrate spectrophotometer
measure O.D of yeast cell culture
H2O wash
LiAc wash
aliquot contents of total competent cell vial

list of pre-conditions:
pre-condition: YPD agar bottle in hot cupboard
pre-condition: yeast vial in -80C deep freeze
pre-condition: sealed yeast colonies plate in cold room
pre-condition: YPD media bottle in cold room
pre-condition: calibrated spectrophotometer
pre-condition: YPD media bottle in non-shaking incubator
pre-condition: yeast culture flask in shaking incubator


materials:
Yeast extract
bacteriological peptone
dextrose
agar no.2
sterile miliQ grade water
para film


Procedure of preparation for experiment: 
preparation of inoculating loop procedure
preparation of conical flask procedure
preparation of sterile container procedure
preparation of frozen yeast vial procedure

experiment procedure:

operating procedure: make YPD agar plates


pre-condition: YPD agar bottle in hot cupboard

experiment action: move
object: petri dish
start location: in store
end location: in laminar flow hood
	
experiment action: move 
object: YPD agar bottle
start location: in hot cupboard
end location: in laminar flow hood
	
experiment action: de-lid
base: petri dish
lid location: in laminar flow hood 

experiment action: pour
component 1: YPD agar
volume: 20ml
precision: +/- 3ml
start container: YPD agar bottle
end container: petri dish 

experiment action: rename
old name:petri dish
name change: YPD agar plate
	
experiment action: wait
goal: to prevent condensation build up
time interval: 30 minutes

experiment action: lid
base: petri dish
lid location: in laminar flow hood

experiment action: join
base: YPD agar plate
top: agar plate lid
goal: prevent drying of YPD agar and maintain sterility
method: method of sealing

experiment action: move 
object: YPD agar plate 
start location: in laminar flow hood
end location: in cold room

post-condition: YPD agar plate in cold room


operating procedure: make single colonies plates

pre-condition: yeast vial in -80C deep freeze

experiment action: move
object: yeast vial
start location: in deep freeze
end location: on ice
equipment: ice machine

experiment action: store
object: yeast vial
goal: storing on ice ensures yeast vial will take longer to thaw
storing condition: ≤4c. 
time point: before frozen yeast vial has completely thawed 

experiment action: move
object: YPD agar plate
start location: in store
end location: in laminar flow hood

experiment action: move
object: inoculating loop
start location: in cold room
end location: in laminar flow hood

experiment action: add
component 1: yeast cells
start container: yeast vial
end container: YPD agar plate
volume: small volume
precision: N/A
equipment: inoculating loop
method: method of inoculating of yeast cells

experiment action: streak
object: yeast cells 
goal: produce single colonies
equipment: inoculating loop
location: in laminar flow hood

experiment action: rename
old name:YPD agar plate
new name:single colonies plate


experiment action: move
object: single colonies plate
start location: in laminar flow hood
end location: in non-shaking incubator

experiment action: incubate
object: yeast single colonies plate
equipment: non-shaking incubator
temperature: 30C
time interval: 24-48h
goal: visible single yeast colonies approximately 2-5mm in diameter

experiment action: join
base: single colonies plate
top: plate lid
goal: prevent drying of YPD agar and maintain sterility
method: method of sealing

experiment action: rename
old name:single colonies plate
new name:sealed yeast colonies plate


experiment action: move
object: sealed yeast colonies plate
start location: in laminar flow hood
end location: in cold room

post-condition: sealed yeast colonies plate in cold room


operating procedure: grow yeast culture

pre-condition: sealed yeast colonies plate in cold room
pre-condition: YPD media bottle in cold room


experiment action: move
object: YPD media bottle
start location: in store
end location: in laminar flow hood

experiment action: move
object: conical flask
start location: in store
end location: in laminar flow hood

experiment action: move 
object: sealed yeast colonies plate   
start location: in cold room
end location : in laminar flow hood

experiment action: add
component 1: YPD medium
volume: 50ml
start container: YPD media bottle
end container: 500ml conical flask
equipment: pipette

experiment action: rename
old name: 500ml conical flask
new name: YPD conical flask

	
experiment action: add 
component 1: single yeast colony
volume: small volume
precision: N/A
start container: yeast single colonies plate
end container: YPD conical flask
equipment: inoculating loop

experiment action: rename
old name:YPD conical flask
new name:yeast culture flask


experiment action: incubate
object: yeast culture flask
equipment: shaking incubator
rpm: 200
temp: 30C
time interval: 12-24h

operating procedure: calibrate spectrophotometer

pre-condition: YPD media bottle in cold room

experiment action: move 
object: cuvette
start location: in store
end location: in laminar flow hood
	
experiment action: move 
object: sterile H2O bottle
start location: in store
end location: in laminar flow hood

experiment action: move 
object: YPD media bottle
start location: in cold room
end location: in laminar flow hood

experiment action: add
component 1: YPD medium
volume: 100microl
equipment: pipette
start container: YPD media bottle
end container: cuvette

experiment action: rename
old name: cuvette
new name: YPD cuvette

experiment action: add
component 1: ddH2O
volume: 900microl
equipment: pipette
start container: sterile H2O bottle
end container: reference cuvette

experiment action: rename
old name: YPD cuvette
new name: reference cuvette


experiment action: mix
object: reference cuvette
method: inversion
time point: until solution is homogeneous

experiment action: move 
object: reference cuvette
start location : in lamina flow hood
end location : in spectrophotometer
	
experiment action: calibrate
object: spectrophotometer
equipment: spectrophotometer
wavelength: 600nm

experiment action: rename
old name: spectrophotometer
new name: calibrated spectrophotometer


experiment action: move 
object: reference cuvette
start location : in calibrated spectrophotometer
end location : on bench

experiment action: remove
component 1: contents of reference cuvette
start location : in reference cuvette
end location : in waste container

post-condition: calibrated spectrophotometer


operating procedure: measure yeast culture optical density   

pre-condition: calibrated spectrophotometer

experiment action: move 
object: yeast culture flask
start location : in shaking incubator
end location : in laminar flow hood

experiment action: move 
object: sterile H2O bottle
start location : in store
end location : in laminar flow hood
	
experiment action: move 
object: cuvette
start location : in store
end location : in laminar flow hood

experiment action: add
component 1: ddH2O
volume: 900microl
equipment: pipette
start container: sterile H2O bottle
end container: cuvette

experiment action: rename
old name: cuvette
new name: H2O cuvette


experiment action: add
component 1: yeast culture
volume: 100microl
equipment: pipette
start container: yeast culture flask
end container: H2O cuvette

experiment action: rename
old name: cuvette
new name: yeast culture cuvette


experiment action: mix
object: yeast culture cuvette
method: inversion
time point: until solution is homogeneous

experiment action: move 
object: yeast culture cuvette
start location : in lamina flow hood
end location : calibrated spectrophotometer

experiment action: measure
object: O.D
wavelength: 600nm
equipment: spectrophotometer

experiment action: record
object: O.D reading
expeced value: 0.2

experiment action: move 
object: yeast culture cuvette
start location : in calibrated spectrophotometer
end location : on bench

experiment action: remove
component 1: contents of yeast culture cuvette
start location : in reference cuvette
end location : in waste container
	
experiment action: calculate 
value: b
formula: dilution factor formula

operating procedure: make yeast culture

pre-condition: YPD media bottle in non-shaking incubator

experiment action: move 
object: 250ml conical flask
start location : in store
end location : in laminar flow hood

experiment action: move 
object: YPD media bottle
start location : in non-shaking incubator
end location : in laminar flow hood

experiment action: move 
object: yeast culture flask
start location : in shaking incubator
end location : in laminar flow hood

experiment action: add
component 1: YPD medium
volume: a-b
equipment: pipette
start container: YPD media bottle
end container: conical flask 

experiment action: rename
old name: conical flask
new name: YPD conical flask


experiment action: add
component 1: yeast culture
volume: b
equipment: pipette
start container: yeast culture flask
end container: YPD conical flask

experiment action: rename
old name: conical flask
new name: yeast competent cell flask


experiment action: move 
object: yeast competent cell flask
start location : in laminar flow 
end location : in shaking incubator

experiment action: incubate
object: yeast competent cell flask
equipment: shaking incubator
rpm: 200
temp: 30C
time: 4h

post-condition: yeast competent cell flask in shaking shaking incubator


operating procedure: calibrate spectrophotometer 

pre-condition: YPD media bottle in cold room

experiment action: move 
object: cuvette
start location : in store
end location : in laminar flow hood
	
experiment action: move 
object: YPD media bottle
start location : in cold room
end location : in laminar flow hood

experiment action: add
component 1: YPD media
volume: 1ml
equipment: pipette
start container: YPD media bottle
end container: cuvette

experiment action: rename
old name: cuvette
new name: YPD reference cuvette


experiment action: mix
object: YPD reference cuvette
method: inversion
time point: until solution is homogeneous

experiment action: move 
object: YPD reference cuvette
start location : in lamina flow hood
end location : in spectrophotometer
	
experiment action: calibrate
object: spectrophotometer
equipment: spectrophotometer

experiment action: rename
old name: spectrophotometer
new name: calibrated spectrophotometer


experiment action: move 
object: YPD reference cuvette
start location : in calibrated spectrophotometer
end location : on bench

experiment action: remove
component 1: contents of YPD reference cuvette
start location : in YPD reference cuvette
end location : in waste container

operating procedure: measure O.D of yeast cell culture

pre-condition: yeast culture flask in shaking incubator

experiment action: move 
object: yeast competent cell flask
start location : in shaking incubator
end location : in laminar flow hood
	
experiment action: move 
object: cuvette
start location : in store
end location : in laminar flow hood

experiment action: add
component 1:  competent cell culture
volume: 1ml
equipment: pipette
start container: yeast competent cell flask
end container: 1ml cuvette

experiment action: rename
old name: cuvette
new name: competent cell cuvette


experiment action: mix
object: competent cell cuvette
method: inversion
time point: until solution is homogeneous

experiment action: move 
object: competent cell cuvette
start location : in lamina flow hood
end location : in spectrophotometer

experiment action: measure
object: O.D
equipment: spectrophotometer
wavelength: 600nm

experiment action: record
object: O.D reading
expected value: 0.6 - 0.8

experiment action: move 
object: yeast culture cuvette
start location : in spectrophotometer
end location : on bench

experiment action: remove
component 1: contents of competent cell cuvette
start location : in competent cell cuvette
end location : in waste container

experiment action
if: O.D>1.5
experiment action: discard
object: competent cell culture 
return to: experiment action make yeast culture

experiment action
if: O.D<0.6
experiment action: move 
object: competent cell culture flask
start location : in laminar flow hood
end location : in shaking incubator

experiment action: incubate
object: competent cell culture flask
equipment: shaking incubator
rpm: 200
temp: 30C
time: 30 mins
comment: an O.D reading of less than 0.4 = cell density is too low. culture must be incubated for longer to allow cell density to reach an o.d =0.6-0.8

experiment action
if: O.D>=0.6 <=1
continue

operating procedure: H2O wash

experiment action: move 
object: 15ml centrifuge tubes
number of objects: 2
start location : in store
end location : in laminar flow hood

experiment action: move 
object: competent cell culture flask
start location : in shaking incubator
end location : in laminar flow hood

experiment action: pour
component 1: competent cell culture
volume: 12ml
start container : in competent cell culture flask
end container : in 15ml centrifuge tube 1

experiment action: rename
old name: 15ml centrifuge tube 1
new name: competent cell tube 1


experiment action: pour
component 1: competent cell culture
volume: 12ml
start container : in competent cell culture flask
end container : in 15ml centrifuge tube 2

experiment action: rename
old name: 15ml centrifuge tube 2
new name: competent cell tube 2
					     
experiment action: move 
object: competent cell tube 1
start location : in laminar flow hood
end location : in bench top centrifuge

experiment action: move 
object: competent cell tube 2
start location : in laminar flow hood
end location : in bench top centrifuge
						
experiment action: centrifuge
object: competent cell tube 1 + 2
rpm: 6000
time: 1min
equipment: bench top centrifuge
				%must this be represented as two actions
experiment action remove
component 1: supernatant
equipment: pipette
start container: competent cell tube 1 
end container: waste container
goal: remove supernatant without removing yeast cells in the process

experiment action remove
component 1: supernatant
equipment: pipette
start container: competent cell tube 2
end container: waste container
goal: remove supernatant without removing yeast cells in the process

experiment action add
component 1: ddH2O
volume: 10ml
equipment: pipette
start container: sterile H2O bottle
end container: competent cell tube 1
	
experiment action add
component 1: ddH2O
volume: 10ml
equipment: pipette
start container: sterile H2O bottle
end container: competent cell tube 2

experiment action: move 
object: competent cell tube 1 
start location : in laminar flow hood
end location : in centrifuge

experiment action: move 
object: competent cell tube 2
start location : in laminar flow hood
end location : in centrifuge

experiment action: centrifuge
object: competent cell tube 1 + 2
rpm: 6000
time: 1mins
equipment: bench top centrifuge

experiment action: move 
object: competent cell tube 1 
start location : in centrifuge
end location : in laminar flow hood

experiment action: move 
object: competent cell tube 2 
start location : in centrifuge
end location : in laminar flow hood

experiment action remove
component 1: supernatant
equipment: pipette
start container: competent cell tube 1
end container: waste container

experiment action remove
component 1: supernatant
equipment: pipette
start container: competent cell tube 2
end container: waste container

experiment action add
component 1: ddH2O
volume: 10ml
equipment: pipette
start container: sterile H2O bottle
end container: competent cell tube 1

experiment action add
component 1: ddH2O
volume: 10ml
equipment: pipette
start container: sterile H2O bottle
end container: competent cell tube 2

experiment action: move 
object: competent cell tube 1
start location : in laminar flow hood
end location : in centrifuge

experiment action: move 
object: competent cell tube 2
start location : in laminar flow hood
end location : in centrifuge

experiment action: centrifuge
object: competent cell tube 1 and 2
rpm: 6000
time: 1mins
equipment: bench top centrifuge

experiment action: move 
object: competent cell tube 1
start location : in centrifuge
end location : in laminar flow hood

experiment action: move 
object: competent cell tube 2
start location : in centrifuge
end location : in laminar flow hood

operating procedure: LiAc wash

experiment action: move 
object: LiAc bottle
number of objects: 1
start location : on bench
end location : in laminar flow hood

experiment action add
component 1: LiAc solution
volume: 10ml
equipment: pipette
start container: liac bottle
end container: competent cell tube 1

experiment action add
component 1: LiAc solution
volume: 10ml
equipment: pipette
start container: liac bottle
end container: competent cell tube 2
	
experiment action: move 
object: competent cell tube 1
start location : in laminar flow hood
end location : in centrifuge

experiment action: move 
object: competent cell tube 2
start location : in laminar flow hood
end location : in centrifuge

experiment action: centrifuge
object: competent cell tube 1 and 2
rpm: 6000
time: 1mins
equipment: bench top centrifuge

experiment action: move 
object: competent cell tube 1
start location : in centrifuge
end location : in laminar flow hood

experiment action: move 
object: competent cell tube 2
start location : in centrifuge
end location : in laminar flow hood

experiment action remove
component 1: supernatant
equipment: pipette
start container: competent cell tube 1
end container: waste container
goal: remove supernatant without removing yeast cells in the process

experiment action remove
component 1: supernatant
equipment: pipette
start container: competent cell tube 2
end container: waste container
goal: remove supernatant without removing yeast cells in the process

experiment action add
component 1: LiAc
volume: 150microl
equipment: pipette
start container: liac bottle
end container: competent cell tube 1 

experiment action resuspend
object: competent cell tube 1
equipment: pipette
time point: keep resspending until solution is homogeneous

experiment action add
component 1: LiAc
volume: 150microl
equipment: pipette
start container: LiAc bottle
end container: competent cell tube 2

experiment action resuspend
object: competent cell tube 2
equipment: pipette
time point: keep resspending until solution is homogeneous

experiment action add
component 1: competent cell LiAc mix
volume: all
equipment: pipette
start container: competent cell tube 2
end container: competent cell tube 1

experiment action add
component 1: competent cell LiAc mix
volume: all
equipment: pipette
start container: competent cell tube 1
end container: 2ml vial

experiment action: rename
old name: 2ml vial
new name: total competent cell vial


operating procedure: aliquot contents of total competent cell vial

experiment action complex: aliquot

experiment action: move 
object: 2ml vial
number of objects: 6
start location : in sterile container
end location : in laminar flow hood

experiment action: label
object: 2ml vial
number of objects: 6
equipment: fine permanent marker

experiment action: rename
old name: 2ml vial
new name: empty labelled 2ml vial


experiment action add
component 1: competent cells
volume: 50microl
equipment: pipette
start container: total competent cell vial
end container: empty labelled 2ml vial

experiment action: rename
old name: empty labelled 2ml vial
new name: labelled competent cell vial


experiment action: move 
object: labelled competent cell vial
number of objects: 6
start location : in lamina flow hood
end location : in -80C deep freeze 
equipment: -80C deep freeze

post-condition: labelled competent cell vial in -80C deep freeze


equipment:
[Wayne: put how many of each where necessary]
vial 	
inoculating loop	1
conical flask		1 
centrifuge tube		2
sterile container	1 
petri dish		2 
bunsen burner		1
laminar flow hood	1
centrifuge		2
bench top centrifuge	1
autoclave		1
spectrophotometer	1
vortex mixer		1
pipette			3
hot cupboard		1
flask			2
beaker			1
jar			2
ice machine		1
-80C deep freeze	1
cold room 		1