Lab meeting 13.03.31  1 st Plasmid preparation of K562, Hela, IM9 -Midi Kit Quiagen  2 nd Prepare control sample within the plasmid preparation process.

Slides:

Advertisements

Similar presentations

Subcloning Techniques

Advertisements

Section G Gene manipulation

09/07/ 24 (yy/mm/dd) Experimenter Kashima, Takashima, Hibino, Miyazawa Note-taker Takashima Title Flow Chart Sample Date Sample NameEnzymepurification.

DNA, Chromosomes By Dr. : Naglaa Mokhtar. DNA Structure.

A Timed Presentation - do not click the mouse. Approximate Run Time - 4 minutes.

Extraction of Nucleic Acids (Genomic DNA, mRNA and Plasmid DNA)

Molecular Biology Working with DNA. Topics  Genomic vs. Vector DNA  Purifying plasmid DNA  Restriction enzymes  Restriction maps.

Mystery Plasmid B ??????. Preliminary Work 18 Possible Plasmids 11 Available Enzymes Too much work… Simplify: 3 Digestions.

A Timed Presentation - do not click the mouse. Approximate Run Time - 4 minutes 5 seconds.

Nucleic Acid Prep Station What we can do for you!.

Plasmid Miniprep. Broad and Long Term Objective To characterize a single clone from an To characterize a single clone from an Emiliania huxleyi cDNA library.

Lab 6 Isolation Techniques

Practical #2: Extraction of genomic DNA from E.Coli Practical #3: Agarose Gel Electrophoresis Bertrand Ong Chan JianPeng Salanne Lee.

Faster, Better Biotech: The rAmylase Project Lab 8g Mini-Prep of pAmylase 2014 Kit Ellyn Daugherty Simon Holdaway.

Chp 3 Purification of DNA from Living Cells Huseyin Tombuloglu PhD. GBE310, Spring 2015.

Week 5 Wednesday: –Ligation of chromosomal digests into plasmid vectors – part II – Checking ligation success Thursday: –Electrocompetent cell preparation.

Isolation of Plasmid DNA June 21, 2007 Leeward Community College.

Mini-Prep Plasmid Isolation and Identification. Page 3-53 in lab manual & handout.

Genomic DNA purification

Molecular Cloning. Overview of Molecular Cloning Restriction digest of plasmid pUC19 and phage –GOAL: Linear pUC19 DNA and several fragments of phage.

Introduction recombinant expression of protein disulfide isomerase (PDI) using the model plant Arabidopsis thaliana Eun Ju Cho ABE workshop 2007.

Abstract Automated, High-Throughput Total and Viral RNA Purification Byung-in Lee 1, Seachol Oak 1, Sharon Khietala 2, Beate Schikora 2, and Karl H Hecker.

Mini-Prep Plasmid Isolation and Identification. Page 3-53 in lab manual & handout.

Molecular Biology Technical Skills. Skills  Micropipetting  Preparing solutions  Working with concentrations  Dilutions  Amounts  Agarose gel electrophoresis.

BIOLOGY 3020 Fall 2008 “Keys of Corn” Project Plasmid isolation Genetic Diversity in corn. Lots of different types of corn are offered for sale at the.

Section G Gene manipulation

Sesperes, John Kenneth Tan, Hannah Michaela Tapia, Paul Adrian Tarriela, Mark Khim Viray, Danielle Grp.5 - HUB42.

Purification of DNA from a cell extract In addition to DNA, bacterial cell wall extract contain significant quantities of protein and RNA. A variety of.

Lab meeting Plasmid preparation and Linearize the pcDNA3.1 vector  pcDNA3.1 digest with ScaI- blunt end  Plasmid preparation using Midi kit.

Select transformants on LB plates containing 100 μg/ml ampicillin  Successful colonies K562: 27 colonies Hela: 11 colonies IM9: 25 colonies Lab Meeting.

From rPERK to myc-tagged mPERK: Construction of pShuttle-loxp- tpA-loxp-mPERK_9E10-IRES- hrGFP-1 plasmid for conditional rescued PERK KO mouse.

Lab meeting Dilute [pcDNA3.1+cDNA U2AF1] up to 20µl (1µg/µl) Linearize pcDNA3.1+cDNA U2AF1 by ScaI ScaI1µl 10X NEB buffer (No.3)5µl BSA2µl.

Plasmids Indispensable tools that allow molecular biologists to obtain essentially unlimited amounts of a DNA sequence Small circular DNA molecules that.

CAPE Biology Workshop on Concepts in Biotechnology & Genetic Engineering Prepared and presented by Dr. Marcia E. Roye.

LET’S PLAY THE REVIEW GAME! (a.k.a how much did you forget this summer?)

Department of Microbiology & Immunology

AYESHA MASRUR KHAN DECEMBER More on Restriction Enzymes 2 Restriction enzymes are Nucleases which can cleave the sugar-phosphate backbone of DNA,

Molecular Tools. Recombinant DNA Restriction enzymes Vectors Ligase and other enzymes.

8.1 - Manipulating & Cloning DNA

Plasmids that contain l cos sites.

A Timed Presentation - do not click the mouse. Approximate Run Time - 2 minutes 38 seconds.

Molecular Biology Working with DNA.

Rec. DNA Lab Wednesday, Jan 24, 2007 Isolation of Chromosomal DNA from Photobacterium leognathi.

Estimation of Nucleic Acid NAHLA BAKHAMIS. 1.Agarose Gel Electrophoresis: Separation and analysing DNA of varying sizes, by moving –ve charge na through.

DNA EXTRACTION Chloe Swinfield.

Lab meeting Nguyen Thi Dai Trang. Electroporation of K562, Hela, IM9  Protocol 1. 2x10 6 cells 2. PBS wash 2 times 3. Suspend in 90µl PBS.

Section II. DNA isolation and analysis 1. Plasmid prep 2. Nucleic acids-RNA, DNA 3. Agarose Gel 4. BioEdit - 소개 Working with DNA- DNA cloning Section III.

Build-a-Gene X Edit. Two projects Traditional method – Using site-directed mutagenesis to edit a gene – Make red fluorescent protein brighter – Weeks.

Restriction Enzyme Digestion of Phage DNA

Restriction digestion DNA concentration Gel electrophoresis

Agarose Gel Electrophoresis of DNA

Extraction and Determination of Bacterial Proteins.

By: Ashneet Biln and Carling Gales

Molecular Therapy - Methods & Clinical Development

From: The role of acetylation in rDNA transcription

MINX RNA nt 120 nt 74 nt 45 nt M M nt 150 nt 239 nt MINX RNA

Fosmid Library Construction Report

Figure 1 tPA and TPO DI and control expression vectors

Purification of DNA from living cells

The common lysis solutions contain A. sodium chloride.

Molecular Biology Working with DNA.

Mini-Prep Plasmid Isolation and Identification

Determine the Identity of Unknown Plasmids

DNA Extraction from Blood

Cloning a DNA segment from bacteriophage

Molecular Therapy - Methods & Clinical Development

Molecular Biology Working with DNA.

RSC Unravels the Nucleosome

Human Argonaute2 Mediates RNA Cleavage Targeted by miRNAs and siRNAs

Presentation transcript:

Lab meeting  1 st Plasmid preparation of K562, Hela, IM9 -Midi Kit Quiagen  2 nd Prepare control sample within the plasmid preparation process Each sample of control was prepared as following: K1: cleared lysate containing supercoiled and open circular plasmid DNA & degraded RNA (240µl) K2: second wash fraction, which ensure that the resin is completely cleared of RNA & other contaminants, leaving only pure plasmid DNA on the column (400µl) K3: the eluate containing pure plasmid DNA with no other contaminating nucleic acids (100µl) Prepare same control sample for Hela (H1, H2, H3) and IM9 (I1, I2, I3)

K1K2K3

Result of control Concentration: K1: 225 ng/µl H1: 178 ng/µl I1: 200 ng/µl K2: 2.7 ng/µl H2: 1.1 ng/µl I2: 3.3 ng/µl K3: 21 ng/µl H3: 20 ng/µl I3: 14 ng/µl A260/280 : A260/230: pcDNA3.1 + cDNA U2AF1: 5989 bp K1 K2 K3 H1 H2 H3 I1 I2 I3 Ladder K562HelaIM9 5000bp 1000bp

Result of plasmid preparation Plasmid formCell lineConcentration (ng/µl) Abs260/Abs280Abs260/Abs230Volume (µl) Open circular & supercoil form [pcDNA3.1+ cDNA U2AF1] K Hela IM

To create stable cell line  Linearize the pcDNA™3.1(+) vector Linearize the pcDNA3.1 + cDNA U2AF1 Source:

pcDNA3.1 digest with ScaI- blunt end

Dilute [pcDNA3.1+cDNA U2AF1] up to 20µl (1µg/µl) Linearize pcDNA3.1+cDNA U2AF1 by ScaI ScaI2µl 10X NEB buffer (No.3)10µl BSA3µl pcDNA3.1+cDNA (1µg/µl)20µl D.W15µl Total 50µl Incubation time: O/N Incubation temp: 37 °C

Plasmid formCell lineConcentration (ng/µl) Abs260/Abs280Abs260/Abs230Volume (µl) K Hela IM Result of plasmid linearization by ScaI Ladder K562 Hela IM9 pcDNA3.1 + cDNA U2AF1: 5989 bp