Progress Report

10/7/14

Projects

• Expression of iGluRs in Xenopus Oocyte• Color Choice Behavior Assay– Generation of Positive Control Lines

DNA Template Preparation for In Vitro Transcription

Template 1 10 ng plasmid DNA

5X Phusion Bu 10 1X

10 mM dNTP 1 200 uM each

M13 Fw (20 uM) 1.25 0.5 uM

M13 Re (20 uM) 1.25 0.5 uM

H2O 35

Phusion (2U/ul) 0.5 0.02U/ul

Total 50

PCR template generation:

Denaturation: 97 CAnnealing: 55 C Extension: 72 C for 3 min# of Cycles: 30

PCR template purified with NuclepSPin Gel and PCR Clean-up Kit (MACHEREY-NAGEL)20 ul elution

Nanodrop:

1 ul for running gel1: CG5621,DKaiRIC, 2902 bps2: CG9935, 3133 bps3: CG3822_DkaiRID, 2995 bps4: CG11155, 3166 bps5: Clumsy, 3442 bps

In Vitro Transcription• Used mMESSAGE mMACHINE® Kit

1(113.5ng/ul) 2 (154 ng/ul) 3 (146 ng/ul) 4 (159 ng/ul) 5 (137.2 ng/ul)

CG5621 CG9935 CG3822 CG11155 clumsy

2X NTP/CAP (ul) 10 10 10 10 10 10X Reaction Buffer (ul) 2 2 2 2 2 0.1–1 μg linear template DNA 4.4 3.2 3.4 3.1 3.6

0.5 ug pcr

Enzyme Mix (ul) 2 2 2 2 2

H2O (ul) 1.6 2.8 2.6 2.9 2.4

RNA sent out for xenopus injectionNanodrop:

RNA Run on Denaturing Agarose Gel

1 2 3 4 5

10 ul for running gel1: CG5621,DKaiRIC, 2902 bps2: CG9935, 3133 bps3: CG3822_DkaiRID, 2995 bps4: CG11155, 3166 bps5: Clumsy, 3442 bps

Color Choice Behavior Assay

-4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0

-0.80

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

G=1uW/cm2

9/16/14

9/11/14

9/17/14

9/19/14

UV/Green Intensity Ratio

PI

CS, equal M & FN=34 for each vial

4 vials, 6-8 days old

7 vials, 5-7 days old

4 vials, 5-7 days old

5 vials, 4-6 days old

Positive Control

Fly Genetics

P:CS

X Tm5C-Gal4 RNAi Clumsy III

+ ; + ; + UAS-Dcr-2 ; ortC1a-G4DB#3#4 OK371dVP16AD ; UAS-RNAi Clumsy III

+ ; + ; + Y Cyo Tm2

F1: UAS-Dcr-2 ; ortC1a-G4DB#3#4 OK371dVP16AD ; UAS-RNAi Clumsy III

+ + +

♂♂

CS & Clumsy III

-4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.80

G=1uw/cm2

CS females

Clumsy III females

4 vials

5 vials

Only Females8 – 10 days oldN=34 for each vial

5 vials

CS & Clumsy III

-4.5 -4 -3.5 -3 -2.5 -2 -1.5

-0.80

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

G=1uw/cm2

Clumsy III females

CS females

Only Females7-9 days oldN=34 for each vial

5 vials

CS Males and Females tested separatelyon 7/30/14

-3.5 -3 -2.5 -2 -1.5 -1 -0.5

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

G=.253uw/cm2

WT males

WT females

UV/Green Intensity Ratio

PI

4 vials

4 vials

N=30 for each vial5-7 days old