Category:						
FL002_hyb_temperature_test						

Contact name:						
Lisa Meadows						

Aim:						
Further test for optimal hyb temperature conditions for FL002 slides using						
male vs female and spike RNA.						

Material:						
Spike mixes FL002 A and FL002 B (made 21/6/05):						
						A:B
r0:	01:01		- 4 spikes		300pg:300pg (on slide)	
r1: 	1.6:1 (8:5)  	- 2 spikes		320pg:200pg		
r2: 	1.8:1 (9:5) 	- 2 spikes		360pg:200pg		
r3: 	02:01	- 2 spikes		400pg:200pg
r4: 	2.2:1 (11:5) 	- 2 spikes		440pg:200pg
r5: 	2.4:1 (12:5) 	- 2 spikes		480pg:200pg

male total RNA (1.20ug/ul - extratced 28/06/05)				
female total RNA (1.53ug/ul - extracted 28/06/05)				

Array type:				
FL002				

Slide numbers:				
S104754				
S104755				
S104756				
S104761				
S104762				
S104763
S104766
S104769
S104771
S104772
S104773
S104776


Protocol:
decided to label the double amount of RNA and spikemix per tube
to save Dye costs.

12 tubes: mixed 100ug male RNA with 40ul SpikeMix A
12 tubes: mixed 100ug female RNA with 40ul SpikeMix B

sped vac and resuspended in 29ul DEPC-water/oligo dT premix

Label as
6 x Cy3: male + A
6 x Cy5: female + B
6 x Cy3: female + B
6 x Cy5: male + A

combined labelling partners and after purifcation pooled
a) Cy3: male + A / Cy5: female + B
b) Cy3: female + B / Cy5: male + A

made 12 aliquots of a and b => 24 slides

froze 12 slides worth after drying in speed vac at -20 degree.

used hyb temperatures:				
47 C				
50 C				


Scanner:				
Axon				
		Cy3-PMT		Cy5-PMT
S104754	550			600
S104755	550			600
S104756	550			600
S104761	550			600
S104762	550			600
S104763	550			600
S104766	550			600
S104769	550			600
S104771	550			600
S104772	550			600
S104773	550			600
S104776	550			600

Results:				

Conclusions:				

Next steps:				

Other comments: