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:				
S104757				
S104759				
S104760				
S104764				
S104768				
S104777


Protocol:
Used same sample as for FL002_hyb_temp_test2 (already labelled and stored at -20C):

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

speed 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:
49 C



Scanner:				
Axon				
		Cy3-PMT		Cy5-PMT
S104757	550			600
S104759	550			600
S104760	550			600
S104764	550			600
S104768	550			600
S104777	550			600

Results:				

Conclusions:				

Next steps:				

Other comments: