Progress Report 10/7/14. Projects Expression of iGluRs in Xenopus Oocyte Color Choice Behavior Assay – Generation of Positive Control Lines.

Slides:

Advertisements

Similar presentations

Polymerase Chain Reaction (PCR)

Advertisements

1 st Strand Synthesis in Reverse Transcription AAAAAAAA 3’ N6 TTT TTTTT 5’ 5’ 3’ Random primer Oligo(dT) primer Sequence specific primer 1 st strand cDNA.

Section J Analysis and application of cloning DNA

COMPUTER EXERCISE Design of PCR and PCR-RFLP experiments This presentation shows all steps of a PCR-RFLP experiment and is a companion of the computer.

Making Nonradioactive Probes: PCR DIG Labelling. Broad Overall Objective Is Myb61 a single or multicopy gene in A. thaliana Is Myb61 a single or multicopy.

Chemical Synthesis, Sequencing, and Amplification of DNA

Analysis of Gene Expression of Arabidopsis using RT-PCR and DNA Cloning Presented by Neha Jain ABE Workshop 2006 June 30, 2006.

General Genetics. PCR 1.Introduce the students to the preparation of the PCR reaction. PCR 2.Examine the PCR products on agarose gel electrophoresis.

DNA Replication DNA mRNA protein transcription translation replication Before each cell division the DNA must be replicated so each daughter cell can get.

1. Sample & Primer CEDDGP001- 카톨릭의대 정의달 * 총 10 개 샘플중에서 4, 14, 24, 28, 36 샘플에 대해서만 PCR 실험을 수행하였다. PCR 실험 수행시 DNA 농도를 100ng/ul 로 희석하여 사용하였다.

Variants of PCR Lecture 4

Progress Report 8/26/14. Projects Color Choice Behavior Assay Expression of iGluRs in Xenopus Oocyte.

Davidson Day Ateh Neuroscience Centre, Institute of Cell and Molecular Sciences Barts and The London School of Medicine and Dentistry Queen Mary University.

Polymerase Chain Reaction

PCR POLYMERASE CHAIN REACTION Dauphin Island Graduate Neurobiology.

EDVOKIT#300: Blue/White Cloning of a DNA Fragment

Genetics Techniques: RFLP & PCR AP Biology Unit 3.

Creating an RNAi feeding vector How does ligation into L4440 work?

Laboratory: Unit 3: purify PCR product (55-65) Lecture: DNA sequencing In-Class Writing: discuss abstracts (pages 68, 157) & AEM 63: , 1997 Hand.

PCR Troubleshooting Virginia Balke

Objective 1 - Describe how restriction enzymes are used to manipulate DNA. Run the virtual gel electrophoresis at this web site

Polymerase Chain Reaction (PCR) Developed in 1983 by Kary Mullis Major breakthrough in Molecular Biology Allows for the amplification of specific DNA fragments.

Molecular Testing and Clinical Diagnosis

PCR is used in; Cloning into plasmid vectors DNA sequencing Genetic screening DNA based phylogeny Functional analysis of genes Identification of DNA fingerprints.

Section J Analysis and application of cloning DNA.

Bo Shi.  Clonings (What I have done.)  Projects (What I am starting to do.)

Amplification of a DNA fragment by Polymerase Chain Reaction (PCR) Ms. Nadia Amara.

PreimmuneMonoclonalanti-Aur-AAffinity Purified anti-Aur-A Blot: retic lysate translation product oocyte extract retic lysate translation.

DNA sequencing. How DNA synthesis takes place inside the living cell?

Today House Keeping Plasmid extraction, EcoRI digest PCR plants 15.ppt Bianca/microarrays (gels)

PCR With PCR it is possible to amplify a single piece of DNA, or a very small number of pieces of DNA, over many cycles, generating millions of copies.

The Polymerase Chain Reaction (PCR)

Introduction to PCR Polymerase Chain Reaction

PUC 19 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ PCR -I pUC 19 specific primers Amplicon purification PCR -II 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Composite pUC 19 primers.

Molecular Cloning. Definitions   Cloning :   Obtaining a piece of DNA from its original source (Genome) and introducing it in a DNA vector   Sub-cloning:

Lab 22 Goals and Objectives: EDVOKIT#300: Blue/White Cloning of a DNA Fragment Calculate transformation efficiencies Compare control efficiency to cloned.

Lecture 4: Polymerase Chain Reaction (PCR)

Restriction Enzyme Store in -20 C Digestion. Restriction Enzyme Buffer NaCl or KCl, TrisHCl, MgCl 2, DTT Different salt: varied activity Buffer supplied.

PCR mediated mutagenesis 2013 년도 2 학기 생화학 실험 (2) 5 주차 조교 : 안성원.

Rajan sharma.  Polymerase chain reaction Is a in vitro method of enzymatic synthesis of specific DNA sequences.  This method was first time developed.

M Gradient PCR using Taq polymerase with 125ng primer concentration M: 1 kb ladder 1: 60°C 2: 62°C 3: 64.4°C 4:66.8°C 5: 69.2°C 6:71.5°C 284D.

I. PCR- Polymerase Chain Reaction A. A method to amplify a specific piece of DNA. DNA polymerase adds complementary strand DNA heated to separate strands.

Topic Cloning and analyzing oxalate degrading enzymes to see if they dissolve kidney stones with Dr. VanWert.

PCR The Polymerase Chain Reaction PCR The Polymerase Chain Reaction.

Introduction to PCR Polymerase Chain Reaction

PCR Basics A review.

Polymerase Chain Reaction (PCR)

L bp 500 bp 250 bp Annealing Temperature in Degrees C V4 16s – 506f-Y/806rB ITS2 – ITS3NGS_mix/ITS4NGS.

EDVOKIT#300: Blue/White Cloning of a DNA Fragment

Polymerase Chain Reaction (PCR)

Overview Wednesday Thursday Labs 12, 13 & 14 due March 7th

Molecular Cloning: Polymerase Chain Reaction

Experimenttal part.

COURSE OF MICROBIOLOGY

Oligose - Primers Qiagen Dneasy Polymerase Chain Reaction What Does it all Mean? Maria Brown October 22, 1009.

cDNA Synthesis Service

Gradient PCR using Taq polymerase with 125ng primer concentration

PCR How does PCR work?: Separation of two strands

Polymerase Chain Reaction (PCR)

3. PCR Page 376 – 377.

Small RNA Sample Preparation

mRNA Sequencing Sample Preparation

Sequencing and Copying DNA

ChIP DNA Sample Preparation

Overview of Recombinant DNA Techniques

Introduction to Polymerase Chain Reaction (PCR)

Restriction Enzyme Digestions

Genomic DNA Sample Preparation

Giorgio Sirugo, Kenneth K. Kidd The American Journal of Human Genetics

The endogenous IGRP gene is selectively expressed in βTC-3 cells and not in Y1 cells. βTC-3 RNA (50 μg) and Y1 RNA (50 μg) were annealed to labeled oligonucleotide.

Presentation transcript:

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 Template1 10 ng plasmid DNA 5X Phusion Bu10 1X 10 mM dNTP1200 uM each M13 Fw (20 uM) uM M13 Re (20 uM) uM H2O35 Phusion (2U/ul) U/ul Total50 PCR template generation: Denaturation: 97 C Annealing: 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 gel 1: CG5621,DKaiRIC, 2902 bps 2: CG9935, 3133 bps 3: CG3822_DkaiRID, 2995 bps 4: CG11155, 3166 bps 5: 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) CG5621CG9935CG3822CG11155clumsy 2X NTP/CAP (ul)10 10X Reaction Buffer (ul) –1 μg linear template DNA ug pcr Enzyme Mix (ul)22222 H2O (ul)

RNA sent out for xenopus injection Nanodrop:

RNA Run on Denaturing Agarose Gel 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

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

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 +++ ♂♂

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

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

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