1. Progress Report 10/7/14

2. Projects Expression of iGluRs in Xenopus Oocyte Color Choice Behavior Assay – Generation of Positive Control Lines

3. DNA Template Preparation for In Vitro Transcription Template1 10 ng plasmid DNA 5X Phusion Bu10 1X 10 mM dNTP1200 uM each M13 Fw (20 uM)1.250.5 uM M13 Re (20 uM)1.250.5 uM H2O35 Phusion (2U/ul)0.50.02U/ul Total50 PCR template generation: Denaturation: 97 C Annealing: 55 C Extension: 72 C for 3 min # of Cycles: 30

4. PCR template purified with NuclepSPin Gel and PCR Clean-up Kit (MACHEREY-NAGEL) 20 ul elution Nanodrop: 1 ul for running gel 1: CG5621,DKaiRIC, 2902 bps 2: CG9935, 3133 bps 3: CG3822_DkaiRID, 2995 bps 4: CG11155, 3166 bps 5: Clumsy, 3442 bps

5. 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) CG5621CG9935CG3822CG11155clumsy 2X NTP/CAP (ul)10 10X Reaction Buffer (ul)22222 0.1–1 μg linear template DNA4.43.23.43.13.6 0.5 ug pcr Enzyme Mix (ul)22222 H2O (ul)1.62.82.62.92.4

6. RNA sent out for xenopus injection Nanodrop:

7. RNA Run on Denaturing Agarose Gel 12345 10 ul for running gel 1: CG5621,DKaiRIC, 2902 bps 2: CG9935, 3133 bps 3: CG3822_DkaiRID, 2995 bps 4: CG11155, 3166 bps 5: Clumsy, 3442 bps

8. Color Choice Behavior Assay CS, equal M & F N=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

9. Positive Control Fly Genetics P: CS X Tm5C-Gal4 RNAi Clumsy III + ; + ; +UAS-Dcr-2 ; ortC1a-G4DB#3#4 OK371dVP16AD ;UAS-RNAi Clumsy III + ; + ; +YCyoTm2 F1:UAS-Dcr-2 ; ortC1a-G4DB#3#4 OK371dVP16AD ;UAS-RNAi Clumsy III +++ ♂♂

10. CS & Clumsy III 4 vials 5 vials Only Females 8 – 10 days old N=34 for each vial

11. 5 vials CS & Clumsy III Only Females 7-9 days old N=34 for each vial 5 vials

12. CS Males and Females tested separately on 7/30/14 4 vials N=30 for each vial 5-7 days old