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Jan 27, 2018

Scientists create DNA robots for future delivering drugs in human body

Scientists have created miniature robots out of DNA that can autonomously "walk" around a surface, pick up certain molecules and drop them off in designated locations, a new study published Thursday in the U.S. journal Science said.
"Just like electromechanical robots are sent off to faraway places, like Mars, we would like to send molecular robots to minuscule places where humans can't go, such as the bloodstream," said LuLu Qian, assistant professor of bioengineering of the California Institute of Technology.
Such technology could one day be used for a wide range of applications, including synthesizing therapeutic chemicals in an artificial molecular factory, delivering drugs in bloodstreams or cells, or sorting molecular components in trash for recycling, Qian said.
To create a DNA robot, Qian's team constructed three basic building blocks, including a "leg" with two "feet" for walking, an "arm" and "hand" for picking up cargo, and a segment that can recognize a specific drop-off point and signal to the hand to release its cargo.
Each of these components is made of just a few nucleotides within a single strand of DNA.
In principle, these modular building blocks could be assembled in many different ways to complete different tasks.
For example, a DNA robot with several hands and arms, could be used to carry multiple molecules simultaneously.
In the work described in the Science paper, the Qian group built a robot that could explore a molecular surface, pick up two different molecules and then distribute them to two distinct regions on the surface.
"The DNA robot moves around on a 58-nanometer-by-58-nanometer pegboard on which the pegs are made of single strands of DNA complementary to the robot's leg and foot," Qian's team said in a statement.
"The robot binds to a peg with its leg and one of its feet -- the other foot floats freely," the team said.
"When random molecular fluctuations cause this free foot to encounter a nearby peg, it pulls the robot to the new peg and its other foot is freed. This process continues with the robot moving in a random direction at each step."
Since one step for the little guy takes five minutes, and allows it to move six nanometers, it may take a day for the robot to explore the entire board, the team said.
"Along the way, as the robot encounters cargo molecules tethered to pegs, it grabs them with its 'hand' components and carries them around until it detects the signal of the drop-off point," it said.
"The process is slow, but it allows for a very simple robot design that utilizes very little chemical energy."
In a summary on the robot, the Science magazine called Qian's work "one small step for a DNA robot, one giant leap for mankind."
"The future is here," the summary wrote.

Posted by DNAworld at 9:51:00 AM 0 comments

Ancient Eurasian DNA sequencing reveals how modern humans migrate

Chinese researchers are discovering unexpected genetic connections between individuals on opposing sides of Eurasia with direct DNA sequencing.
Their study suggests a complex history that may represent early gene flow across Eurasia or an early population structure that eventually led to Europeans and Asians.
In a review published in the journal Trends in Genetics on Thursday, scientists at the Chinese Academy of Sciences in Beijing discuss the genetics of ancient individuals from Europe and Western Asia between 45,000 to 7,500 years ago.
The authors summarized work that investigated the genomes of more than 70 ancients in the Eurasian family tree.
"Aside from these individuals, it is a fact that sampling for the Eurasian region is sparse for all time periods except the present-day," says the correspondent author Fu Qiaomei, a paleogeneticist at the Chinese Academy of Sciences.
"But with the information from the several individuals available for ancient DNA sequencing we do have hints at interesting population structure, migration and interaction in East Asia."
The researchers learned that in Eurasia between 35,000 and 45,000 years ago, at least four distinct populations were present. These were early Asian and Europeans, as well as populations with ancestry hardly found or not at all in modern populations.
It shows that, by 7,500 to 14,000 years ago, the populations across Eurasia shared genetic similarities, suggesting greater interactions between geographically distant populations.
These analyses also revealed at least two Neanderthal population mixing events, one approximately 50,000 to 60,000 years ago and a second more than 37,000 years ago.
Fu and colleagues hope to extend this type of sequencing and analysis to learn more about the genetic prehistory of East Asia and other regions, including Oceania, Africa, and the Americas.
"All of those areas have a rich human prehistory, particularly in Africa, so any ancient DNA from those continents will likely resolve some major questions on human migration," she says.

Posted by DNAworld at 9:51:00 AM 0 comments

DNA Modification/Epigenetics

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

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
Abstract  Full Text  Full Text (PDF)

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
Abstract  Full Text  Full Text (PDF)

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
Abstract  Full Text  Full Text (PDF)

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
Abstract  Full Text  Full Text (PDF)

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
Abstract  Full Text  Full Text (PDF)

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
Abstract  Full Text  Full Text (PDF)

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

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

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
Abstract  Full Text  Full Text (PDF)

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

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
Abstract  Full Text  Full Text (PDF)

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

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
Abstract  Full Text  Full Text (PDF)

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
Abstract  Full Text  Full Text (PDF)

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

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
Abstract  Full Text  Full Text (PDF)

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

Purification of Recombinant Drosophila ACF
Craig L. Peterson
Cold Spring Harb Protoc 2009; doi:10.1101/pdb.prot5115
Abstract  Full Text  Full Text (PDF)

Purification of Recombinant Drosophila NAP1
Craig L. Peterson
Cold Spring Harb Protoc 2009; doi:10.1101/pdb.prot5116
Abstract  Full Text  Full Text (PDF)

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
Abstract  Full Text  Full Text (PDF)

Chromosome Conformation Capture
Nathan F. Cope and Peter Fraser
Cold Spring Harb Protoc 2009; doi:10.1101/pdb.prot5137
Abstract  Full Text  Full Text (PDF)

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

Salt Gradient Dialysis Reconstitution of Nucleosomes
Craig L. Peterson
Cold Spring Harb Protoc 2008; doi:10.1101/pdb.prot5113
Abstract  Full Text  Full Text (PDF)

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
Abstract  Full Text  Full Text (PDF)

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
Abstract  Full Text  Full Text (PDF)

Mapping Protein Distributions on Polytene Chromosomes by Immunostaining
Renato Paro
Cold Spring Harb Protoc 2008; doi:10.1101/pdb.prot4714
Abstract  Full Text  Full Text (PDF)

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
Abstract  Full Text  Full Text (PDF)

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
Abstract  Full Text  Full Text (PDF)

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
Abstract  Full Text  Full Text (PDF)

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
Abstract  Full Text  Full Text (PDF)

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
Abstract  Full Text

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
Abstract  Full Text

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
Abstract  Full Text

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
Abstract  Full Text

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

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

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
Abstract  Full Text

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

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


Posted by DNAworld at 9:46:00 AM 0 comments

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.


Posted by DNAworld at 9:45:00 AM 0 comments

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.

Epigenome NoE - Protocols Database
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 

Protocols- Epigenetics and Methylation Protocols > Bisulfite ...
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/

DNA Methylation in Cancer - Protocols - Southern Blot Analysis ...
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=...

DNA methylation analyis using restriction enzyme digestion
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 -

EpiTYPER for Quantitative DNA Methylation Analysis | Protocol ...
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

Epigenome NoE - protocol: DNA Methylation Analysis by Bisulfite ...
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

CSH Protocols -- Collected Res9
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

Epigenetics- Top Hits
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  

Nature Protocols: DNA methylation analysis by pyrosequencing
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

Immunolabelling, immunolabeling and immunostaining of proteins in ...
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  

DNA Methylation in Cancer - Protocols - DNA-Methyltransferase ...
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 School. 1400 University Ave ...
www.genomecenter.ucdavis.edu/farnham/protocols/MOJO%20Dec%202003%20ChIP-chip%20protocol.pdf

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

DNA Methylation Analysis Using Restriction Enzyme Digestion
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 -

DNA methylation analysis using bisulphite sequencing
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 -

DNA methylation analysis using Single Nucleotide Primer Extension (SNuPE)
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 -

Cancer Genetics in the 21st Century - National Cancer Institute (Video)
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


Posted by DNAworld at 9:44:00 AM 0 comments

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/

Posted by DNAworld at 9:44:00 AM 0 comments

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）

Posted by DNAworld at 8:35:00 PM 0 comments

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