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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
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