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Jan 27, 2018

DNA Modification/Epigenetics

Analysis of Mitotic Checkpoint Function in Xenopus Egg Extracts
Yinghui Mao
Cold Spring Harb Protoc 2018; doi:10.1101/pdb.prot099853

Analysis of DNA Methylation in Mammalian Cells
Paul M. Lizardi, Qin Yan, and Narendra Wajapeyee
Cold Spring Harb Protoc 2017; doi:10.1101/pdb.top094821

Methylation-Specific Polymerase Chain Reaction (PCR) for Gene-Specific DNA Methylation Detection Paul M. Lizardi, Qin Yan, and Narendra Wajapeyee
Cold Spring Harb Protoc 2017; doi:10.1101/pdb.prot094847

Methyl-Cytosine-Based Immunoprecipitation for DNA Methylation Analysis
Paul M. Lizardi, Qin Yan, and Narendra Wajapeyee
Cold Spring Harb Protoc 2017; doi:10.1101/pdb.prot094854

High-Throughput Deep Sequencing for Mapping Mammalian DNA Methylation
Paul M. Lizardi, Qin Yan, and Narendra Wajapeyee
Cold Spring Harb Protoc 2017; doi:10.1101/pdb.prot094862

DNA Bisulfite Sequencing for Single-Nucleotide-Resolution DNA Methylation Detection
Paul M. Lizardi, Qin Yan, and Narendra Wajapeyee
Cold Spring Harb Protoc 2017; doi:10.1101/pdb.prot094839

Illumina Sequencing of Bisulfite-Converted DNA Libraries
Paul M. Lizardi, Qin Yan, and Narendra Wajapeyee
Cold Spring Harb Protoc 2017; doi:10.1101/pdb.prot094870

Micrococcal Nuclease Digestion of Schizosaccharomyces pombe Chromatin
Hugh P. Cam and Simon Whitehall
Cold Spring Harb Protoc 2016; doi:10.1101/pdb.prot091538

Oncogenomics Methods and Resources
Simon J. Furney, Gunes Gundem, and Nuria Lopez-Bigas
Cold Spring Harb Protoc 2012; doi:10.1101/pdb.top069229

Detection of Cytosine Methylation in RNA Using Bisulfite Sequencing
Tim Pollex, Katharina Hanna, and Matthias Schaefer
Cold Spring Harb Protoc 2010; doi:10.1101/pdb.prot5505

In Vitro Histone Demethylase Assay
Yu-ichi Tsukada and Keiichi I. Nakayama
Cold Spring Harb Protoc 2010; doi:10.1101/pdb.prot5512

Potassium Permanganate Probing of Pol II Open Complexes
Michael F. Carey, Craig L. Peterson, and Stephen T. Smale
Cold Spring Harb Protoc 2010; doi:10.1101/pdb.prot5479

Amplification of Bisulfite-Converted DNA for Genome-Wide DNA Methylation Profiling Jon Reinders
Cold Spring Harb Protoc 2009; doi:10.1101/pdb.prot5342

In Vivo DNase I, MNase, and Restriction Enzyme Footprinting via Ligation-Mediated Polymerase Chain Reaction (LM-PCR)
Michael F. Carey, Craig L. Peterson, and Stephen T. Smale
Cold Spring Harb Protoc 2009; doi:10.1101/pdb.prot5277

In Vivo Dimethyl Sulfate (DMS) Footprinting via Ligation-Mediated Polymerase Chain Reaction (LM-PCR) Michael F. Carey, Craig L. Peterson, and Stephen T. Smale
Cold Spring Harb Protoc 2009; doi:10.1101/pdb.prot5278

Chromatin Immunoprecipitation (ChIP)
Michael F. Carey, Craig L. Peterson, and Stephen T. Smale
Cold Spring Harb Protoc 2009; doi:10.1101/pdb.prot5279

Native Chromatin Preparation and Illumina/Solexa Library Construction
Suresh Cuddapah, Artem Barski, Kairong Cui, Dustin E. Schones, Zhibin Wang, Gang Wei, and Keji Zhao
Cold Spring Harb Protoc 2009; doi:10.1101/pdb.prot5237

Reconstitution of Nucleosomal Arrays Using Recombinant Drosophila ACF and NAP1Craig L. Peterson
Cold Spring Harb Protoc 2009; doi:10.1101/pdb.prot5114

Purification of Recombinant Drosophila ACF
Craig L. Peterson
Cold Spring Harb Protoc 2009; doi:10.1101/pdb.prot5115

Purification of Recombinant Drosophila NAP1
Craig L. Peterson
Cold Spring Harb Protoc 2009; doi:10.1101/pdb.prot5116

Combined 3C-ChIP-Cloning (6C) Assay: A Tool to Unravel Protein-Mediated Genome Architecture
Vijay K. Tiwari and Stephen B. Baylin
Cold Spring Harb Protoc 2009; doi:10.1101/pdb.prot5168

Chromosome Conformation Capture
Nathan F. Cope and Peter Fraser
Cold Spring Harb Protoc 2009; doi:10.1101/pdb.prot5137

Chicken Erythrocyte Histone Octamer Preparation
Craig L. Peterson and Jeffrey C. Hansen
Cold Spring Harb Protoc 2008; doi:10.1101/pdb.prot5112

Salt Gradient Dialysis Reconstitution of Nucleosomes
Craig L. Peterson
Cold Spring Harb Protoc 2008; doi:10.1101/pdb.prot5113

DNA Methylation Analysis of Human Imprinted Loci by Bisulfite Genomic Sequencing
Vanessa T. Angeles and Renee A. Reijo Pera
Cold Spring Harb Protoc 2008; doi:10.1101/pdb.prot5046

Coimmunoprecipitation (co-IP) of Nuclear Proteins and Chromatin Immunoprecipitation (ChIP) from Arabidopsis
Berthe Katrine Fiil, Jin-Long Qiu, Klaus Petersen, Morten Petersen, and John Mundy
Cold Spring Harb Protoc 2008; doi:10.1101/pdb.prot5049

Mapping Protein Distributions on Polytene Chromosomes by Immunostaining
Renato Paro
Cold Spring Harb Protoc 2008; doi:10.1101/pdb.prot4714

DNA Immunoprecipitation (DIP) for the Determination of DNA-Binding Specificity
Andrea J. Gossett and Jason D. Lieb
Cold Spring Harb Protoc 2008; doi:10.1101/pdb.prot4972

Methylated CpG Island Amplification and Microarray (MCAM) for High-Throughput Analysis of DNA Methylation
Marcos R. H. Estécio, Pearlly S. Yan, Tim H-M. Huang, and Jean-Pierre J. Issa
Cold Spring Harb Protoc 2008; doi:10.1101/pdb.prot4974

In Vitro Histone Methyltransferase Assay
Ian M. Fingerman, Hai-Ning Du, and Scott D. Briggs
Cold Spring Harb Protoc 2008; doi:10.1101/pdb.prot4939

Development of Mammalian Cell Lines with lac Operator-Tagged Chromosomes
Yuri G. Strukov and Andrew S. Belmont
Cold Spring Harb Protoc 2008; doi:10.1101/pdb.prot4903

Micrococcal Nuclease-Southern Blot Assay: I. MNase and Restriction Digestions
Michael Carey and Stephen T. Smale
Cold Spring Harb Protoc 2007; doi:10.1101/pdb.prot4890

Micrococcal Nuclease-Southern Blot Assay: II. Capillary Transfer and Hybridization
Michael Carey and Stephen T. Smale
Cold Spring Harb Protoc 2007; doi:10.1101/pdb.prot4891

Chromatin Immunoprecipitation (ChIP) on Unfixed Chromatin from Cells and Tissues to Analyze Histone Modifications
Alexandre Wagschal, Katia Delaval, Maëlle Pannetier, Philippe Arnaud, and Robert Feil
Cold Spring Harb Protoc 2007; doi:10.1101/pdb.prot4767

PCR-Based Analysis of Immunoprecipitated Chromatin
Alexandre Wagschal, Katia Delaval, Maëlle Pannetier, Philippe Arnaud, and Robert Feil
Cold Spring Harb Protoc 2007; doi:10.1101/pdb.prot4768

Yeast Chromatin Immunoprecipitation (ChIP) Assay
William P. Tansey
Cold Spring Harb Protoc 2007; doi:10.1101/pdb.prot4642

Denaturing Protein Immunoprecipitation from Yeast
William P. Tansey
Cold Spring Harb Protoc 2007; doi:10.1101/pdb.prot4643

Chromatin Immunoprecipitation (ChIP) of Protein Complexes: Mapping of Genomic Targets of Nuclear Proteins in Cultured Cells
Achim Breilingand Valerio Orlando
Cold Spring Harb Protoc 2006; doi:10.1101/pdb.prot4560

Mapping DNase-I-hypersensitive Sites
Joseph Sambrook and David W. Russell
Cold Spring Harb Protoc 2006; doi:10.1101/pdb.prot3949

Chromatin Immunoprecipitation in Yeast
David C. Amberg, Daniel J. Burke, and Jeffrey N. Strathern
Cold Spring Harb Protoc 2006; doi:10.1101/pdb.prot4177


Gene therapy

Gene therapy involves supplying a functional gene to cells lacking that function, with the aim of correcting a genetic disorder or acquired disease. Gene therapy can be broadly divided into two categories. The first is alteration of germ cells, that is, sperm or eggs, which results in a permanent genetic change for the whole organism and subsequent generations. This “germ line gene therapy” is considered by many to be unethical in human beings. The second type of gene therapy, “somatic cell gene therapy”, is analogous to an organ transplant. In this case, one or more specific tissues are targeted by direct treatment or by removal of the tissue, addition of the therapeutic gene or genes in the laboratory, and return of the treated cells to the patient. Clinical trials of somatic cell gene therapy began in the late 1990s, mostly for the treatment of cancers and blood, liver, and lung disorders.

Despite a great deal of publicity and promises, the history of human gene therapy has been characterized by relatively limited success. The effect of introducing a gene into cells often promotes only partial and/or transient relief from the symptoms of the disease being treated. Some gene therapy trial patients have suffered adverse consequences of the treatment itself, including deaths. In some cases, the adverse effects result from disruption of essential genes within the patient's genome by insertional inactivation. In others, viral vectors used for gene therapy have been contaminated with infectious virus. Nevertheless, gene therapy is still held to be a promising future area of medicine, and is an area where there is a significant level of research and development activity.


Epigenetics Protocols

Background of Epigenetics:
There is far more to genetics than the sequence of building blocks in the DNA molecules that make up our genes and chromosomes. The "more" is known as epigenetics.

What is epigenetics?
Epigenetics, literally "on" genes, refers to all modifications to genes other than changes in the DNA sequence itself. Epigenetic modifications include addition of molecules, like methyl groups, to the DNA backbone. Adding these groups changes the appearance and structure of DNA, altering how a gene can interact with important interpreting (transcribing) molecules in the cell's nucleus.

How do epigenetic modifications affect genes?
Genes carry the blueprints to make proteins in the cell. The DNA sequence of a gene is transcribed into RNA, which is then translated into the sequence of a protein. Every cell in the body has the same genetic information; what makes cells, tissues and organs different is that different sets of genes are turned on or expressed.

Epigenetics Protocols Database. The following protocols are selected, solicited and ... Protocol Online �� Epigenetic Station's Top Hits �� Cold Spring Harbor ...
www.epigenome-noe.net/researchtools/protocols.php 

Bisulfite Treatment Methylation Assay Protocol. Bisulfite Methylation. ... or epigenetic information differs on the two complementary strands of DNA. ...
www.molecularstation.com/.../Bisulfite_Treatment_Methylation_Assay_Protocol/

Care Centers & Departments, --, Care Center Information, A to Z Department List, --, Brain & SpineCenter, Breast Center ...
www.mdanderson.org/departments/methylation/display.cfm?id=816A773B-F17B-43D4-9B0090D858EED92B&method=...

This site contains protocols, product information and other material for molecular biologists, biochemists, immunologists, and all people interested in ...
www.methods.info/Methods/DNA_methylation/Restriction_analysis.html -

Protocol Archive. BioTechniques Online ... Epigenetics. Epigenetic Modification Analysis ... Return to table of contents for the Protocol Guide ...
www.biotechniques.com/default.asp?page=protocol&subsection=article_display&id=112658

Genomic DNA methylation is one of the most important epigenetic modifications in eukaryotes. It is essential for life and its alteration is often associated ...
www.epigenetics-noe.net/researchtools/protocol.php?protid=34

DNA Modification/Epigenetics. Protocols 1-10 of 13 total displayed. ... Protocol Icon. DNA Immunoprecipitation (DIP) for the Determination of DNA-Binding ...
www.cshprotocols.org/cgi/collection/dna_modification_epigenetics

Chromosome Conformation Capture (3C) Protocol developed by Alice Horton .... The Epigenetics Journal publishes original research and reviews covering ...
epigeneticstation.com/epigenetic-links/index.php?list=top  

This Protocol is listed in the following Categories: Gene expression. Author(s): J?rg Tost Affiliation(s): Laboratory for Epigenetics, CEA-Institut de ...
www.natureprotocols.com/2007/09/06/dna_methylation_analysis_by_py.php

Science. Retroelements �� Pararetrovirus �� Bio banana Musa genomics �� Microarrays �� Repetitive DNA �� Chromosome Model �� Bovid (cow) diversity and evolution ...
www.le.ac.uk/bl/phh4/immunopr.htm  

Care Centers & Departments, --, Care Center Information, A to Z Department List, -, Brain & SpineCenter, Breast Center ...
www.mdanderson.org/departments/methylation/display.cfm?id=9B5B6082-C53A-4A86-BEE3EE149A52251E&method=...

Matthew J. Oberley, Peggy J. Farnham McArdle Laboratory for Cancer ...
Matthew J. Oberley, Peggy J. Farnham. 1. McArdle Laboratory for Cancer Research. University of Wisconsin Medical School1400 University Ave ...
www.genomecenter.ucdavis.edu/farnham/protocols/MOJO%20Dec%202003%20ChIP-chip%20protocol.pdf

A listing of protocols in Drosophila (fruit fly) research available on the Web.
www.ceolas.org/VL/fly/protocols.html  

This site contains protocols, product information and other material for molecular biologists, biochemists, immunologists, and all people interested in ...
www.methods.info/Methods/DNA_methylation/Restriction_analysis.html -

This site contains protocols, product information and other material for molecular biologists, biochemists, immunologists, and all people interested in ...
www.methods.info/Methods/DNA_methylation/Bisulphite_sequencing.html -

This site contains protocols, product information and other material for molecular biologists, biochemists, immunologists, and all people interested in ...
www.methods.info/Methods/DNA_methylation/SNuPE.html -

Between these Dates:. Jan. Feb. Mar. Apr. May, Jun. Jul. Aug. Sept. Oct. Nov. Dec. 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008 ...
www.nci.nih.gov/newscenter/benchmarks-vol5-issue1/Video


Inoutscripts.com search engine script does not work at all


I have purchased InOutScripts search engine for my new project and have to say, their support is terrible, their software does not work and I have been scammed once again by people who just take money for nothing. They claim that it uses FREE APIs, NONE OF THE APIS ARE FREE, unless you limit your website users to allow them only 100 Google searches a month and 5000 Bing, Yahoo charges no matter what now. Also, I purchased the translator plugin, this does not work without a paid API.

I also purchased the Google theme but nothing works. They say that they offer free installation, but their lazy staff Rojin P Mani, said rudely "I am too busy to do this, install it yourself!" DISGUSTING!!!

The Google theme is full of errors and when I report these errors Rojin P Mani (who seems to be the only person running the site) said "Sorry I can not get to the office today".

They have very lazy staff who do not care once you have paid them.

The script will only return results from Google and not Bing.

Amazon does not work. eBay does not work, either.

$872 waste of money! For this price for a script, I think that after 7 days, I should have something that works!!!

AVOID INOUTSCRIPTS AT ALL COSTS, THEY DO NOT CARE AFTER YOU HAVE PAID THEM.


This phishing scamming group members include Jacob, Kumar, Nair, Saranya, and more

If you are a victim of Inoutscripts, feel free to contact me. Let's bring these criminals into justice together.
https://forums.digitalpoint.com/threads/inoutscripts-search-engine-script-my-honest-review-terrible.2596611/

Sep 16, 2016

DNA MICROARRAY PROTOCOL

i)           Set-up the following Pre-Hybridisation solution in a Coplin Jar and        incubate at 65°C during the labeling incubation period to equilibrate. 20X SSC 8.75 ml 20% SDS 0.25 ml BSA (100 mg/ml) 5.0 ml H2O to 50.0 ml

ii)            Label control and test genomic DNA as follows:- CONTROL TEST Genomic DNA ˜ 2 mg ˜ 2 mg Random Hexamers (3 mg/ml) 1 ml 1 ml H2O to 41.5 ml to 41.5 ml Heat at 95ºC for 5 minutes. Snap cool on ice and briefly centrifuge. 10X buffer 5 ml 5 ml dNTP's (5mM each dATP, dGTP & dTTP, 2mM dCTP) 1 ml 1 ml Cy-labelled dCTP 1.5 ml (Cy3) 1.5 ml (Cy5) Klenow fragment (10U/ml) 1 ml 1 ml Incubate at 37°C for 90 minutes.

iii)          Incubate the microarray slide(s) in the Pre-Hybridisation solution for 20 minutes at 65°C, beginning just before the end of the labelling reactions incubation time at 37°C.

iv)          Combine the control and test reactions and purify using the Qiagen MinElute PCR Purification kit, using a two step wash stage using 500 ml then 250 ml volumes of Buffer PE and eluting the labeled cDNA from the MinElute column with 14 ml H2O. The columns retain approximately 1 ml, so the final eluted volume will be 13 ml.

v)           Rinse the pre-hybridised microarray slides in H2O for 1 minute, then in isopropanol for 1 minute. Spin at 1500 rpm for 5 minutes to dry slides. Keep in covered slide box. 1 NICK DORRELL - LAST UPDATE FEBRUARY 2004

vi)          Prepare the Hybridisation solution as follows: - Sample 13 ml H2O 26 ml 20X SSC 12 ml 2% SDS 9 ml Heat at 95ºC for 2 minutes. Allow to cool slowly at room temperature and centrifuge for 30 seconds. Add 2 x 20 ml H2O to the corners of the hybridisation chamber. Place a slide into the chamber. Place a LifterSlip™ glass coverslip (22 mm x 25 mm) over the array section on the slide using tweezers. Pipette the Hybridisation solution onto the slide at the top of the coverslip. Seal the chamber and incubate in a water bath at 65°C overnight.

vii)         Prepare Wash solutions as follows: - Wash A (1X SSC 0.5% SDS) Wash B (0.06X SSC) 20X SSC 20 ml 2.4 ml 20% SDS 1 ml H2O to 400 ml to 800 ml Incubate Wash A solution at 65ºC overnight. Dispense 400 ml volumes into three glass slide washing dishes. Remove slide(s) from the hybridisation chambers and gently remove coverslip(s) by rinsing in Wash A. Place slide(s) in a slide rack and rinse with agitation for 5 minutes. Transfer slide(s) to a clean slide rack and rinse with agitation in Wash B(i) for 2 minutes, then in Wash B (ii) for a further 2 minutes. Spin at 1500 rpm for 5 minutes to dry slide(s).

viii)      Scan slide(s) using Affymetrix 418 scanner and analyse data


NICK DORRELL - LAST UPDATE FEBRUARY 2004

Polymerase Chain Reaction (PCR) Analysis

PCR analysis is a technique that allows technicians to create millions of precise DNA replications from a single sample of DNA. In fact, DNA amplification alongside PCR can let forensic scientists perform DNA analysis on samples that are as tiny as only a couple of skin cells. In contrast to some other DNA analysis techniques, PCR analysis has the advantage of analysing minuscule sample sizes, even if they are degraded although they must not be contaminated with DNA from other sources during the collection, storage and transport of the sample.

Restriction Fragment Length Polymorphism (RFLP)

RFLP is a technique that is not widely used now but it was one of the first techniques used for DNA analysis in forensic science. Large sample sizes are needed for RFLP relative to newer techniques - usually a sample would need to be approximately the size of a one-pound coin. While that in itself may sound small, it is large relative to other techniques such as PCR analysis that require only a few cells for successful sequencing. In RFLP, the different lengths of DNA fragments are analysed. These fragments are from the digestion of a sample of DNA with a restriction endonuclease enzyme. The enzyme chops DNA in a certain style - the restriction endonuclease recognition site. Whether or not particular recognition sites are present will provide different lengths of DNA fragments, which are then divided up through electrophoresis. DNA probes then serve to hybridise the fragments through complementary binding.