miRNA Proposed Experiments

L7ae

EXPERIMENTS

Hef1a eBFP2 & Hef1a 2x Kturn eYFP (positive control)
1. Should be yellow and blue
Hef1a eBFP2 & Hef1a 2x Kturn eYFP & Hef1a-L7ae (negative control)
1. Should be blue (minimal yellow)

TAL14

Name	Location	Concentration
pEXPR: Hef1a-eBFP2	Our -20	Currently being propagated
pEXPR: Hef1a-Gal4Vp16	With Jin	Will meet with Jin
pEXPR: Hef1a-TAL14	With Brian	pENTR will be LR'd with Hef1a when obtained
pEXPR: 5xUAS/4xTAL14bs-eYFP	With Samira	UAS needs to be added somehow

EXPERIMENTS:

Hef1a-eBFP2 & 5xUAS/4xTAL14bs-eYFP
1. negative control (minimal yellow)
Hef1a-eBFP2 & Hef1a-Gal4VP16 & 5xUAS/4xTAL14bs-eYFP
1. positive control (hopefully linear BFP/YFP)
Hef1a-eBFP2 & Hef1a-Gal4VP16 & Hef1a-TAL14 & 5xUAS/4xTAL14bs-eYFP
1. Should be blue (minimal yellow)

n-WELL PLATE PROPORTIONS:

STEP	COMPONENT	6-well	24-well	96-well
1.	Adherent Cells	0.25-1 x 10^{^6}	0.5-2 x 10^{^5}	1-4 x 10^{^4}
3.	Opti-MEM Medium	150uL x 4	50uL	25uL x 4
3.	Lipofectamine LTX Reagent	6, 9, 12, 15 uL	1.5uL	1, 1.5, 2, 2.5 uL
4.	Opti-MEM Medium	700uL	250uL	125uL
	DNA (0.5-5 ug/uL)	14ug	5ug	2.5ug
	PLUS Reagent	14uL	5uL	2.5uL
5.	Diluted DNA (with PLUS)	150uL	50uL	25uL
5.	Diluted Lipofectamine LTX	150uL	50uL	25uL
7.	DNA-lipid complex (per well)	250uL	50uL	10uL
7.	FINAL DNA (PER WELL)**	2500ng	500ng	100ng

Plasmid (Concentration)	6-well	24-well	96-well
pEXPR hEF1A:eBFP2 (96 ng/ul)	13 ul (2 plasmids) 8.7 ul (3 plasmids) 6.5 ul (4 plasmids)	2.6 ul (2 plasmids) 1.8 ul (3 plasmids) 1.3 ul (4 plasmids)	.5 ul (2 plasmids) .4 ul (3 plasmids) .3 ul (4 plasmids)
pEXPR hEF1A:l7ae (186 ng/ul)	4.5 ul (3 plasmids)	.9 ul (3 plasmids)	-