Category:
FL002_hyb_temperature_test

Contact name:
Bettina Fischer

Aim:
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:	1:1		- 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: 	2:1          	- 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:
S104722
S104726
S104728
S104729
S104732
S104736
S104737
S104739
S104740
S104741
S104743
S104745
S104730
S104742
S104738
S104723
S104724
S104725
S104744
S104731
S104733
S104727
S104734
S104735

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:
52 C
54 C
50 C
56 C


Scanner:
Axon
		Cy3-PMT		Cy5-PMT
S104722	550			600
S104726	550			600
S104728	550			600
S104729	550			600
S104732	550			600
S104736	550			600
S104737	550			600
S104739	550			600
S104740	550			600
S104741	550			600
S104743	550			600
S104745	550			600
S104730	550			600
S104742	550			600
S104738	550			600
S104723	550			600
S104724	550			600
S104725	550			600
S104744	550			600
S104731	550			600
S104733	550			600
S104727	550			600
S104734	550			600
S104735	550			600

Results:

Conclusions:

Next steps:

Other comments: