Prepare 0.5 M EDTA

To prepare EDTA at 0.5 M (pH 8.0): Add 186.1 g of disodium EDTA-2H2O
to 800 ml of H2O. Adjust the pH to 8.0 with NaOH.

Chromosome conformation capture (3C) analysis

[DOC]

Chromosome conformation capture (3C) analysis

File Format: Microsoft Word - View as HTML
To generate control templates for the positive controls, BAC clones were used for all the loci of interest. For the TH2 locus we used the BAC clone B182 ...
www.nature.com/nature/journal/v435/n7042/extref/nature03574-s6.doc -

Random Shear BAC Library Construction

[PDF]

Random Shear BAC Library Construction

File Format: PDF/Adobe Acrobat - View as HTML
The quality of genomic BAC libraries depends greatly on the cloning methods and vectors .... BAC Cloning System simplifies BAC preparation and sequencing ...
www.lucigen.com/catalog/images/pdfs/newsletters/Random_ShearBAC.pdf -

How the CopyControl™ BAC Cloning Kits Work

[PDF]

How the CopyControl™ BAC Cloning Kits Work

File Format: PDF/Adobe Acrobat - View as HTML
The CopyControl BAC Cloning Kits–. based on technology developed in the. laboratory of Dr. Waclaw Szybalski. 1. at the University of Wisconsin-Madison ...
www.epibio.com/pdfforum/9_3ccbacclone.pdf

Protocol for CopyControl’ BAC Cloning Kit

[PDF]

Protocol for CopyControl' BAC Cloning Kit

File Format: PDF/Adobe Acrobat - View as HTML
CopyControl™ Products including, but not limited to, the CopyControl BAC Cloning Kit are covered by U.S. Patent No. 5874259 licensed to ...
www.epibio.com/pdftechlit/177pl055.pdf

Dynamic Building of a BAC Clone Tiling Path

Dynamic Building of a BAC Clone Tiling Path for the Rat Genome ...

the sequence of each BAC clone is generated by assembling the ... genome is obtained by merging overlapping BAC clone se-. quences. ...
www.genome.org/cgi/reprint/14/4/679.pdf

Direct Cloning and Sequencing of Bacterial Artificial Chromosome

Direct Cloning and Sequencing of Bacterial Artificial Chromosome ...

of BAC clone inserts is an essential step in chromosome walking and map- .... sites, one from the BAC cloning site and the other from the pBlueScriptII ...
www.springerlink.com/index/G0U66PK677681N2K.pdf

BAC-CLONING AND MUTAGENESIS

BAC-CLONING AND MUTAGENESIS OF HERPESVIRUS GENOMES

Cloning as an infectious plasmid and mutagenesis in E.coli has become the method of choice for genetic analysis of viruses with small genomes. Until recently, this approch was not accessible for herpesviruses since their large genomes (150 to 230 kbp) could not be cloned in one piece in E.coli....

Read Full Article Here

http://www.lmb.uni-muenchen.de/groups/messerle/bac.htm

BAC Clone Ordering

Only genome sequences determined by systematic bacterial artificial chromosome (BAC) clone sequencing will have clones available. Whole Genome Shotgun (WGS) clones are generally not available for ordering, but if the genome sequences are the result of a composite assembly, BAC clones may be available in certain regions. The human genome sequence however has been solely determined by systematic BAC clone sequencing...

Read full article here

http://www.ensembl.org/info/data/docs/clone_ordering.html

How to identify an address

How to identify an address:

The membrane is divided into 6 fields (diagram provided). Each field contains 384 squares. The 384 squares represent the row and column identification of the BAC. Within each square there are 16 positions where 8 clones are spotted in duplicate (diagram). The pattern of the spotted clones will generate the plate address of the BAC. To identify your clone, please follow the directions below.

The most complicated part about identifying a clone address is that consecutive plates are not spotted into each field. The 384 well plates are spotted onto the membrane with plates 1-6 spotted into fields 1-6 respectively (duplication pattern 1, see diagram). Since there is a total of 6 fields on the membrane, the cycle will continue with the next six consecutive plates (plates 7 through 12) again being spotted into fields 1 through 6 respectively, but in a different duplication pattern (duplication p.....

Full Protocols Here

http://www.genome.clemson.edu/groups/bac/protocols/addressnew.html

Megabase-size DNA isolation from plants

Megabase-size DNA isolation from plants

To construct large insert DNA libraries in BAC and YAC vectors, methods must be developed to isolate very high molecular weight DNA - megabase-size DNA - from plants. To isolate such DNA, protoplasts or nuclei must first be embedded in agarose plugs or microbeads. The agarose acts as a solid yet porous matrix which allows for the diffusion of various reagents for DNA purification and subsequent manipulations while preventing the DNA from being sheared (Schwartz and Cantor, 1984). Microbeads are preferred over plugs because the use of beads increases the surface area surrounding the tissue sample by approximately 1000 fold thereby allowing for more efficient and rapid diffusion of chemicals and enzymes into and out of the agarose beads (Cook, 1984, Overhauser and Radic, 1987, Wing..........

Read Full Protocol Here
http://www.genome.clemson.edu/groups/bac/protocols/megabasedna.html

Clemson BAC Filter Manual

Clemson BAC Filter Manual

The purpose of this manual is to explain what the filters are, clone designation procedure, what screening conditions to use, and clone analysis for the Clemson University Arabidopsis BAC library. Filter users are urged to send clone information to schoi@clemson.edu for deposition into the BAC web depository.

I. What are the filters

You have received 3 Hybond N+ filt.......

http://www.genome.clemson.edu/groups/bac/protocols/bacmanual.html

Bacterial artificial chromosome (BAC) libraries

The construction of bacterial artificial chromosome (BAC) libraries

SANGDUN CHOI 1,2 and ROD A. WING 2

1 Department of Soil and Crop Sciences, Texas A&M University, College Station, TX 77843, USA

2 Departments of Agronomy & Biological Sciences, Clemson University Genome Center, Clemson University, Clemson, SC 29634, USA

Introduction

Cloning of exogenous DNA into bacterial artificial chromosomes (BACs) provides a new approach to the analysis of the genomes of higher organisms [1]. BAC libraries containing large genomic DNA inserts are important tools for positional cloning, physical mapping and genome sequencing. A number of human and plant BAC libraries have been constructed (e.g., human: [2], Arabidopsis: [3], rice: [4], sorghum: [5]). Bacterial artificial chromosome vectors utilize the E. coli single-copy fertility plasmid and can maintain genomic DNA fragments up to 350 kb. Very little or no rearrangement of the inserts or chimerism have been observed [1, 5, 6, 7]. Other systems for the cloning of large DNA fragments have been developed. The development of yeast artificial chromosome vectors (YAC: [8]) permits cloning of fragme

Full Protocol Here

http://www.genome.clemson.edu/groups/bac/protocols/protocols2new.html

Growing up of BAC DNA clones

Growing up of BAC clones

1. BACs are usually supplied as stabs in agar (2.5 ml screw-top tubes filled with LB agar, stabbed with a toothpick which has been put into a bacterial colony). These are then frozen; at -70C they will keep indefinitely, at -20C for a year or more, at 4C for several months and at room temperature (e.g. during shipping) for a week.

2. Make up 10 ml LB broth + 3 ul of chloramphenicol (dissolved in alcohol, 50 mg/ml, so final concentration is 15 ug/ml, stored at 4C) for each stab which will be grown up....

Read Full Protocol Here

http://www.le.ac.uk/biology/phh4/bacclone.htm

Mapping & PCR

[PPT]

Mapping & PCR

File Format: Microsoft Powerpoint - View as HTML
GeneScan (ABI). PCR related technologies. Primer extension .... problems are minimized since both the order and the amount of overlap of reads are known ...
www.med.nyu.edu/rcr/rcr/course/PPT/map-seq.ppt -

Mutagenesis PPT

[PPT]

Mutagenesis

File Format: Microsoft Powerpoint - View as HTML
Site-directed Mutagenesis by PCR. Site-specific Mutagenesis by Overlap Extension. Xylanases Analyzed in This Study. CmXyn10B periplasmic xylanase produced ...
163.23.209.240/micro/Biotech/93/Mutagenesis.ppt -

Splice Overlap Extension (SOE) Protocol

[DOC]

Splice Overlap Extension (SOE)

File Format: Microsoft Word - View as HTML
Splice Overlap Extension (SOE). SOE utilizes the PCR thermal cycler to anneal two DNA fragments with partially overlapping complementary 3' ends ...
web.ics.purdue.edu/.../Work_CSS/Data%20Files/Protocols_MTPL/General%20MB/Splice%20Overlap%20Extension.doc

Introduction of Multiple Mutations Using Overlap Extension PCR

Simultaneous Introduction of Multiple Mutations Using Overlap Extension PCR

BioTechniques 22:28-30 (January 1997)

Introduction of multiple mutations can be accomplished through phage
M13-based site-directed mutagenesis using several oligonucleotides (6). The
major drawback of this method is the low efficiency of generating all the in-
tended mutations simultaneously (7,9). The alternative methods for introducing
multisite mutations are based on poly-merase chain reaction (PCR) (1)

Full Details Here

[PDF]

Benchmarks

File Format: PDF/Adobe Acrobat - View as HTML

PCR mutagenesis techniques and RTS

PCR mutagenesis techniques and RTS

File Format: PDF/Adobe Acrobat - View as HTML ates the linear expression template via overlap extension PCR, .... Figure 14: Scheme of domain fusion by overlap extension PCR using the RTS E. coli Linear ... www.roche-applied-science.com/sis/proteinexpression/literature/manual/chapter1/RTS_30_35.pdf

Asymmetric overlap extension PCR method

Asymmetric overlap extension PCR method bypassing intermediate ...

PCR and overlap extension. Lanes 1 and 2 contain the. asymmetric PCR products (RBca-5F and .... Mehta RK, Singh J (1999) Bridge-overlap-extension PCR ...
www.springerlink.com/index/BX8225317K76458T.pdf

Overlap extension PCR method

A modified overlap extension PCR method to create chimeric genes ...

In conclusion, a modified overlap extension PCR technique. is described to improve the construction of multiple. chimeric genes and proteins, site-directed ... www.springerlink.com/index/LWN2032286220T71.pdf -

Overlap extension PCR

Overlap extension PCR

This type of PCR is used to make mutations, fuse two gene segments together, make insertions within a gene, or make deletions within a gene.

Set up the two half reactions as follows:

Vent Polymerase Reactions for the two halves:

• 80.5 ul distilled H₂O
• 10 ul ThermoPol buffer
• 2 ul 10 mM dNTPs
• 2 ul 100 mM MgSO₄
• 2 pmoles primerA or B, usually 2 ul of a 100 pM stock
• 2 pmoles primerC or D, usually 2 ul of a 100 pM stock
• 1 ul miniprep or Qiagen DNA, usually around 200-500 ng template
• 0.5 ml Vent DNA Polymerase

Note: Volume of distilled H₂O can be adjusted if additional volumes of reagents are required.

PCR Cycle (for PE 9600 or equivalent):..

Read Full Protocol here

http://www.gardnerlab.org/protocols/oePCR.htm

Gardner Lab Research: Overlap Extension PCR

cDNA synthesis by overlap extension PCR

Biotechniques. 2004 Jul;37(1):124, 126, 128-9.

Aptamer-dependent full-length cDNA synthesis by overlap extension PCR.

Mitani Y, Nakayama T, Harbers M , Hayashizaki Y .

RIKEN Yokohama Institute, Yokohama, Japan.

Sequencing of the human genome in combination with computational annotation has provided tremendous data on predicted genes. However, for most of them, no corresponding cDNAs are available yet. Furthermore, even where cDNA clones were obtained, gene transcripts often have many different splice variants that are not covered by current gene collections. For direct synthesis of cDNA clones corresponding to predicted genes, new splice variants, or any other gene of interest, we established optimal PCR conditions for the direct amplification of exons from genomic DNA, which require a specific Taq aptamer. PCR products comprising differently tagged exons were concatenated by overlap extension into full-length cDNAs. To prove the effectiveness of the approach, the 1900-bp full-length open reading frame of the human mitochondrial aldehyde dehydrogenase (ALDH2) gene was synthesized in a two-step reaction comprising all 13 exons. Thus, our conditions are of general value for in vitro synthesis of cDNAs and alternative splice variants from genomic DNA.

PMID: 15283210 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&list_uids=15283210&cmd=Retrieve&indexed=google

Mutagenesis by PCR-driven overlap extension

Gene splicing and mutagenesis by PCR-driven overlap extension ...

Nature Protocols is an interactive online resource for all laboratory protocols relevant to biological and biomedical research. Nature Protocols includes a ...
www.nature.com/nprot/journal/v2/n4/full/nprot.2007.132.html

Polymerase Chain Reaction (PCR) Method

Polymerase Chain Reaction (PCR) Method That Fulfills All of the Consensus Recommendations for the Use of PCR in the Diagnosis of Bordetella pertussis Infections.

FARRELL DJ, MCKEON M, DAGGARD G, MUKKUR TK.

Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 1999 Sep 26-29; 39: 225 (abstract no. 1569).

Queensland Health Pathology Service, Toowoomba, AUSTRALIA

BACKGROUND: Although many PCR methods have been reported, none has yet fulfilled the consensus recommendations for the use of PCR in the diagnosis of B. pertussis (BP) infections (Meade & Bollen, J Med Microbiol 1994; 41:51) and hence a standardised method has not been forthcoming. The aim of this study was to develop such a method.METHODS: A rapid cycle PCR method with a microwell format/probe hybridisation detection step was developed using novel oligonucleotides targeted at the pertussis toxin operon. An internal control was developed using overlap extension PCR and mouse beta-actin DNA. A novel hybridisation stringency ratio (HSR) was developed to differentiate BP from B. parapertussis (BPP) and B. bronchiseptica (BB). Contamination was minimised by the use of heat labile uracil N-glycosylase. Specimens were treated by simply diluting 1:10 in H[2]O and then heating for 20 minutes at 99.9[o]C. Analytical sensitivity and specificity were ascertained using 35 strains of Bordetella species and 30 strains of common respiratory pathogens and commensal organisms. Clinical sensitivity and specificity were ascertained using 79 specimens tested using a nested PCR method targeting the insertion sequence IS481 (Farrell et al., J Clin Microbiol 1999; 37:606).RESULTS: Analytical specificity was 100%. Analytical sensitivity was comparable to nested IS481 PCR (1 organism per reaction). 35/36 nested PCR positive specimens were positive - inhibitory substances being detected in the remaining specimen (which became positive after DNA purification). 2 specimens that fulfilled a clinical definition of pertussis were positive by the new method/negative by IS481 PCR. 41 specimens were negative by both methods. All Bordetella species and clinical specimens were identified correctly to the species level using HSR.CONCLUSION: The following were successfully developed: 1) A PCR method which fulfils all recommendations with the added benefit of being rapid (4 hours) and reproducible. 2) A novel, simple mechanism (HSR) to allow differentiation between BP and BPP/BB post-amplification.

Full Details Here

http://gateway.nlm.nih.gov/MeetingAbstracts/102245122.html

Gene splicing by overlap extension

Biotechniques. 1990 May;8(5):528-35.Links

Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction.

Horton RM, Cai ZL , Ho SN, Pease LR.

Dept. of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905.

Gene Splicing by Overlap Extension or "gene SOEing" is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. By modifying the sequences incorporated into the 5'-ends of the primers, any pair of polymerase chain reaction products can be made to share a common sequence at one end. Under polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another, forming an overlap. Extension of this overlap by DNA polymerase yields a recombinant molecule. This powerful and technically simple approach offers many advantages over conventional approaches for manipulating gene sequences.

PMID: 2357375 [PubMed - indexed for MEDLINE]

Site-directed mutagenesis by overlap extension using the polymerase chain reaction

Gene. 1989 Apr 15;77 (1):51-9 2744487 [Cited: 762]
Site-directed mutagenesis by overlap extension using the polymerase chain reaction.
[My paper] S N Ho , H D Hunt , R M Horton , J K Pullen , L R Pease
Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends. These fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand. The resulting fusion product is amplified further by PCR. Specific alterations in the nucleotide (nt) sequence can be introduced by incorporating nucleotide changes into the overlapping oligo primers. Using this technique of site-directed mutagenesis, three variants of a mouse major histocompatibility complex class-I gene have been generated, cloned and analyzed. Screening of mutant clones revealed at least a 98% efficiency of mutagenesis. All clones sequenced contained the desired mutations, and a low frequency of random substitution estimated to occur at approx. 1 in 4000 nt was detected. This method represents a significant improvement over standard methods of site-directed mutagenesis because it is much faster, simpler and approaches 100% efficiency in the generation of mutant product.
Mesh-terms: Amino Acid Sequence; Animals; DNA, Recombinant; DNA-Directed DNA Polymerase; Exons; Gene Amplification; Genes, MHC Class I;Genes, Synthetic; Genetic Engineering, methods; Mice; Molecular Sequence Data; Mutation; Oligodeoxyribonucleotides, chemical synthesis; Oligodeoxyribonucleotides, genetics; Support, Non-U.S. Gov't; Support, U.S. Gov't, P.H.S.; Taq Polymerase;
[Pubmed] [Scholar] [EndNote] [BibTex] http://lib.bioinfo.pl/pmid:2744487

Overlap extension polymerase chain reaction

From Wikipedia, the free encyclopedia

The overlap extension polymerase chain reaction (OE-PCR) technique was first published in 1988 by Higuchi et al. and describes a process based on the polymerase chain reaction used to a) insert mutations at specific points in a sequence, further than ~55 nucleotides from either end and/or b) produce polynucleotides from smaller fragments.<sup>[1]

Full Details Here

http://en.wikipedia.org/wiki/Overlap_extension_polymerase_chain_reaction</sup>

Mutagenesis by PCR Protocols (CSH)

	Rapid and Efficient Site-directed Mutagenesis by the Single-tube Megaprimer PCR Method Joseph Sambrook and David W. Russell CSH Protocols; 2006; doi:10.1101/pdb.prot3467 [Extract] [Full text]
	Site-specific Mutagenesis by Overlap Extension Joseph Sambrook and David W. Russell CSH Protocols; 2006; doi:10.1101/pdb.prot3468 [Extract] [Full text]
	In Vitro Mutagenesis Using Double-stranded DNA Templates: Selection of Mutants with DpnI Joseph Sambrook and David W. Russell CSH Protocols; 2006; doi:10.1101/pdb.prot3813 [Extract] [Full text]
	Genetic Engineering with PCR Joseph Sambrook and David W. Russell CSH Protocols; 2006; doi:10.1101/pdb.prot3836 [Extract] [Full text]
	Mutagenic PCR R. Craig Cadwell and Gerald F. Joyce CSH Protocols; 2006; doi:10.1101/pdb.prot4143 [Extract] [Full text]
	Rapid PCR Site-Directed Mutagenesis Gina L. Costa and Michael P. Weiner CSH Protocols; 2006; doi:10.1101/pdb.prot4144 [Extract] [Full text]
	PCR-Mediated Gene Disruption: One-Step Method David C. Amberg, Daniel J. Burke, and Jeffrey N. Strathern CSH Protocols; 2006; doi:10.1101/pdb.prot4169 [Extract] [Full text]

Purification for PCR CSH Protocols

	Purification of Genomic DNA from Whole Blood: A Solution-Based Method Craig Smith, Paul Otto, Rex Bitner, and Gary Shiels CSH Protocols; 2006; doi:10.1101/pdb.prot4096 [Extract] [Full text]
	A Silica Membrane-Based Method for the Isolation of Genomic DNA from Tissues and Cultured Cells Craig Smith, Paul Otto, Rex Bitner, and Gary Shiels CSH Protocols; 2006; doi:10.1101/pdb.prot4097 [Extract] [Full text]
	DNA IQ Isolation of Genomic DNA from Stains and Buccal Swabs Craig Smith, Paul Otto, Rex Bitner, and Gary Shiels CSH Protocols; 2006; doi:10.1101/pdb.prot4098 [Extract] [Full text]

Polymerase Chain Reaction PCR

PCR

Polymerase Chain Reaction

1) Add the following to a microfuge tube:
10 ul reaction buffer
1 ul 15 uM forward primer
1 ul 15 uM reverse primer
1 ul template DNA
5 ul 2 mM dNTP
8 ul 25 mM MgCl2 or MgSO4 (volume variable)
water (to make up......

Full Details Here

http://www.biochem.ucl.ac.uk/bsm/nmr/protocols/protocols/PCR.html

Detection of Alu by PCR

A Human DNA Fingerprinting Lab Protocol

1994 Cold Spring Harbor Laboratory
DNA Learning Center

In this experiment, polymerase chain reaction (PCR) is used to amplify a nucleotide sequence from chromosome 8 to look for an insertion of a short DNA sequence called Alu within the tissue plasminogen activator (TPA) gene. Although the DNA from different individuals is more alike than different, there are many regions of the human chromosomes t..

Full Details Here

http://www.accessexcellence.com/AE/AEPC/DNA/detection.html

Calculating Concentrations for PCR

Molecular Biology Techniques Manual

Third Edition

Edited by:

Vernon E Coyne, M Diane James, Sharon J Reid and Edward P Rybicki

Primers

Nucleotides

http://www.mcb.uct.ac.za//pcrconcn.htm

Protocol for PCR with Taq DNA Polymerase

Protocol for PCR with Taq DNA Polymerase

Avoiding Contamination

PCR allows the production of more than 10 million copies of a target DNA sequence from only a few molecules. The sensitivity of this technique means that the sample should not be contaminated with any other DNA or previously amplified products (amplicons) that may reside in the laboratory environment.

Full Protocol Here

http://www.fermentas.com/techinfo/pcr/dnaamplprotocol.htm

PCR Amplification of DNA

PCR Amplification of DNA

Materials:

	sterile water
	10X amplification buffer with 15mM MgCl2
	10 mM dNTP
	50 μM oligonucleotide primer 1
	50 μM oligonucleotide primer 2
	5 unit/μl Taq Polymerase
	template DNA (1 μg genomic DNA, 0.1-1 ng plasmid DNA) in 10 μl
	mineral oil (for thermocyclers without a heated lid 1. Combine the following for each reaction (on ice) in a 0.2 or 0.5 ml tube:  10X PCR buffer 10 μl Primer 1 1 μl Primer 2  1 μl dNTP  2 μl template DNA and water 85.5 μl Taq Polymerase 0.5 μl 2. Prepare a control reaction with no template DNA and an additional 10 μl of sterile water. 3. If the thermocycler does not have a heated lid, add 70-100 μl mineral oil (or 2 drops of silicone oil) to each reaction. 4. Place tubes in a thermal cycler preheated to 94 degrees C. 5. Run the following program:

94 degrees C 1 min

55 degrees C 1 min or annealing temperature appropriate for particular primer pair

72 degrees C 1 min (if product is <500 bp), 3 min (if product is >500 bp)

for 30 cycles.

Program a final extension at 72 degrees C for 7 min.

Interactions of Blood Cell Constituents

Interactions of Blood Cell Constituents: Experimental investigation and Computational Modeling by Discrete Particle Dynamics Algorithm
Microvascular Research, In Press, Accepted Manuscript, Available online 12 October 2007
N. Filipovic, D. Ravnic, M. Kojic, S.J. Mentzer, S. Haber and A. Tsuda

Computer model to forecast gyrodactylid infections on fish hosts

Gyroscope: an individual-based computer model to forecast gyrodactylid infections on fish hosts
International Journal for Parasitology, In Press, Accepted Manuscript, Available online 14 October 2007
C. van Oosterhout, R. Potter, H. Wright and J. Cable

Multivariate Analysis

Identifying functional imaging markers of mild cognitive impairment in early Alzheimer’s and Parkinson’s disease using multivariate analysis
Clinical Neuroscience Research, Volume 6, Issue 6, November 2007, Pages 367-373
Chaorui Huang, Paul Mattis and Per Julin

Ethical issues in withholding and withdrawing life-prolonging medical treatment

Ethical issues in withholding and withdrawing life-prolonging medical treatment in the ICU
Current Anaesthesia & Critical Care, In Press, Uncorrected Proof, Available online 11 October 2007
Margot Heaney, Carole Foot, William D. Freeman and John Fraser

Care of the brain-dead organ donor

Care of the brain-dead organ donor
Current Anaesthesia & Critical Care, In Press, Uncorrected Proof, Available online 11 October 2007
Konstantinos Linos, John Fraser, William D. Freeman and Carole Foot

Orthotopic liver transplantation

Orthotopic liver transplantation in Jehovah's Witnesses
Current Anaesthesia & Critical Care, In Press, Uncorrected Proof, Available online 15 October 2007
Lucy Yang and Zorica Jankovic

Fluorescence In Situ Hybridization

[DOC]

Fluorescence In Situ Hybridization

File Format: Microsoft Word - View as HTML Among these are techniques like primed in situ (PRINS) labeling or FISH with ... The probes are then PCR-labeled through the incorporation of either ... www.aecom.yu.edu/home/hgp/GIF%20techniques.doc

In situ hybridization protocol using Dig-labelled probes

[DOC]

Kellogg Lab in situ hybridization protocol using Dig-labelled probes

File Format: Microsoft Word - View as HTML In situ hybridization 1: Tissue Fixation, dehydration and embedding, .... Purify the PCR reaction using RNA only silica spin columns (i.e. not the kits that ... www.csulb.edu/~smalcomb/documents/Simon_in_situ.doc

In situ hybridization

[DOC]

In situ hybridization

File Format: Microsoft Word - View as HTML In situ hybridization. Brains were removed immediately after decapitation, ... Each predicted RT-PCR product spanned an intron/exon junction. ... www.nature.com/nature/journal/v421/n6925/extref/nature01388-s1.doc -

In-situ Protocol for larval/pupal/adult tissues

[DOC]

In-situ Protocol for larval/pupal/adult tissues

File Format: Microsoft Word - View as HTML Supplemental Protocol for RNA in situ hybridization in fly larval/pupal/adult ... Probes are PCR amplified and cloned into the Topo vector for verification. ... www.biomedcentral.com/content/supplementary/1749-8104-2-14-S7.doc

RT-PCR, in situ, immunohistochemistry

[DOC]

RT-PCR, in situ, immunohistochemistry

File Format: Microsoft Word - View as HTML We recommend the use of an unequivocal nomenclature to refer to in situ hybridization (Pv), RT-PCR (PvRT) and immunocytochemical (PV) data. ... www.columbia.edu/.../faculty/yuste/petilla/petilla-webpages/classification/first%20draft/MolecularClassi.doc -

Localized in Situ Amplification (LISA)

Localized in Situ Amplification (LISA): A Novel Approach to in ...

in situ PCR. protocol. and. is detected. by a nonra-. dioactive. immunohistochemical ..... by in situ hybridiza-. tion. after PCR-ampliflcation. Am J Pathol ... www.clinchem.org/cgi/reprint/40/3/381.pdf -

In Situ Hybridisation: Theory and Applications

[PDF]

In Situ Hybridisation: Theory and Applications

File Format: PDF/Adobe Acrobat - View as HTML DIRECT In situ PCR. Labeled PCR. Reaction Mix. Tissue attached to. microscope slides ... In situ PCR: Examples. Human papilloma virus type 16 in ... www.surrey.ac.uk/SBMS/ACADEMICS_homepage/plant_nick/Downloads/MSc%20Lectures/Appl%20Tox_ISH.pdf

In situ PCR Technique based on pricking microinjection for cDNA

In situ PCR Technique based on pricking microinjection for cDNA ...

In situ cDNA synthesis and PCR were conducted by pricking pathogen-invaded epidermal cells (HES) or appressoria (FIS) of the pow- ... www.springerlink.com/index/LYWP8031LWNE7TM9.pdf -

In situ polymerase chain reaction: toy or tool?

In situ polymerase chain reaction: toy or tool?

rect in situ PCR techniques utilizing a single pair of. primers, most probably due to the ... situ PCR reaction must be monitored by several reliable ... www.springerlink.com/index/M534142784438042.pdf

In situ PCR: protocols and applications

In situ PCR: protocols and applications.

groups have published protocols and data using in situ PCR techniques. (5). This article will discuss some of the basic concepts of in situ PCR, the proto- ... www.genome.org/cgi/reprint/4/4/S151.pdf -

Agricultural Biotechnology × In Situ PCR Techniques

[PDF]

Agricultural Biotechnology × In Situ PCR Techniques ×

File Format: PDF/Adobe Acrobat - View as HTML agriculture, not adequately covered in most textbooks available. Emili Montesinos. University of Girona. In Situ PCR Techniques ... www.im.microbios.org/05march99/14%20Book%20reviews.PDF

A protocol for PCR in situ hybridization of hyphomycetes

[PDF]

A protocol for PCR in situ hybridization of hyphomycetes

File Format: PDF/Adobe Acrobat - View as HTML The variables for in situ hybridization and for PCR were ... Key words Hyphomycetes · In situ hybridization · In situ PCR · Rock inhabiting ... www.im.microbios.org/03setember98/08%20Sterflinger.pdf -

In Situ Reverse Transcription-PCR for Monitoring Gene Expression

In Situ Reverse Transcription-PCR for Monitoring Gene Expression in Individual Methanosarcina mazei …

M Lange, T Tolker-Nielsen, S Molin, BK Ahring - Applied and Environmental Microbiology, 2000 - Am Soc Microbiol
... In situ PCR procedure. A GeneAmp EZ rTth RNA PCR kit ... Detection of in situ
PCR products. Two different systems were used to detect ...
Cited by 17

In situ PCR. An overview

In situ PCR. An overview

AA Long, P Komminoth - Methods Mol Biol, 1997 - neurosci.humanapress.com
... In Situ PCR: An Overview. PRINS and In Situ PCR Protocols September 1996
pps. 141-162 ISBN: Series: Methods in Molecular Biology ...
Cited by 15

In situ PCR: pathologist's dream or nightmare?

In situ PCR: pathologist's dream or nightmare?

JJ O'Leary, R Chetty, AK Graham, JO McGee - J Pathol, 1996 - ncbi.nlm.nih.gov
In situ PCR: pathologist's dream or nightmare? O'Leary JJ, Chetty R, Graham AK,
McGee JO. Nuffield Department of Pathology and Bacteriology ...
Cited by 21

Flow cytometric detection of specific gene expression in prokaryotic cells using in situ RT-PCR

Flow cytometric detection of specific gene expression in prokaryotic cells using in situ RT-PCR

FM Letters - FEMS Microbiology Letters, 2000 - Blackwell Synergy
... detect the possession of a specific functional gene and its expression by amplifying
DNA and mRNA targets inside bacterial cells with in situ PCR technologies. ...
Cited by 17

In-situ immuno-PCR to detect antigens

In-situ immuno-PCR to detect antigens

Y Cao, K Kopplow, GY Liu - The Lancet, 2000 - Elsevier
... In-situ immuno-PCR to detect antigens. ... In-situ immunoassays do not allow the detection
of the minute numbers of target molecules accessible with in-situ PCR. ...
Cited by 15

In situ localization of PCR-amplified human and viral cDNAs

In situ localization of PCR-amplified human and viral cDNAs

GJ Nuovo, GA Gorgone, P MacConnell, M Margiotta, … - Genome Research, 1992 - Cold Spring Harbor Lab
... We describe a technique, called reverse transcriptase (RT) in situ PCR, whereby
RNA may be nonisotopically detected in fixed cells when amplified by PCR after ...
Cited by 19

Pitfalls of in situ polymerase chain reaction (PCR)

Pitfalls of in situ polymerase chain reaction (PCR) using direct incorporation of labelled … -

JF Sallstrom, I Zehbe, M Alemi, E Wilander - Anticancer Res, 1993 - ncbi.nlm.nih.gov
... False positivity is reported of in situ PCR reactions in a direct incorporation
assay with digoxigenin-labelled dUTP. It is recommended ...
Cited by 28

IN SITU PCR AMPLIFICATION SYSTEM

IN SITU PCR AMPLIFICATION SYSTEM -

LA HAFF, JG ATWOOD - 1993 - patents1.ic.gc.ca
... ABSTRACT: Abstract of the Disclosure A complete in situ PCR system for amplification
of nucleic acids contained in a prepared cell or tissue sample. ...
Cited by 35

In-situ polymerase chain reaction

In-situ polymerase chain reaction. An overview of methods, applications and limitations of a new … -

P Komminoth, AA Long - Virchows Arch B Cell Pathol Incl Mol Pathol, 1993 - ncbi.nlm.nih.gov
... The in-situ polymerase chain reaction (in-situ PCR) is a novel molecular technique
that combines the extreme sensitivity of the PCR with the anatomical ...
Cited by 83

Direct In Situ PCR Method for Detection of Specific Bacteria

Development of a Direct In Situ PCR Method for Detection of Specific Bacteria in Natural … -

K Tani, K Kurokawa, M Nasu - Applied and Environmental Microbiology, 1998 - Am Soc Microbiol
... Development of a Direct In Situ PCR Method for Detection of Specific Bacteria in
Natural Environments. ... The ECOL primer was used in direct in situ PCR. ...
Cited by 54 -

In situ DNA PCR and RNA hybridization detection of herpes simplex virus

In situ DNA PCR and RNA hybridization detection of herpes simplex virus sequences in trigeminal … -

A Mehta, J Maggioncalda, O Bagasra, S Thikkavarapu … - Virology, 1995 - ncbi.nlm.nih.gov
... of latently infected mice was detected by an in situ DNA polymerase chain reaction
(PCR), which includes a DNA:DNA hybridization step (indirect in situ PCR). ...
Cited by 52 -

Reverse Transcriptase In Situ PCR Technique

Reverse Transcriptase In Situ PCR Technique Source CSH Protocols; 2006; doi:10.1101/pdb.prot4119 Overview This protocol describes a method for reverse transcriptase (RT) in situ PCR. In situ PCR differs from PCR in situ hybridization in the inclusion of a reporter molecule in the amplification step. The two steps of RT in situ PCR that differ from in situ PCR are overnight digestion in RNase-free DNase that is performed after protease digestion, and an RT step, prior to in situ PCR. By Gerard J. Nuovo.

http://www.biocompare.com/jumpres.asp?type=protocol&protocolid=2441&siteid=30

In situ PCR

In situ PCR for detection and identification of fungal species

To overcome this problem, we designed an in situ PCR technique that links PCR ... first report of in situ PCR on phytopathogenic fungal material. ... journals.cambridge.org/production/action/cjoGetFulltext?fulltextid=110878

In situ PCR Expression Analysis

FGP: in situ PCR Expression Analysis

Method: in situ PCR expression analysis (method details to be added). There have been 10374 hits since 09/26/2003. Please note: suggested browsers are ... www.floralgenome.org/fgp/methods/method_in_situ.html - 10k - Cached

PCR In Situ Hybridization: Protocols and Applications

Archives of Pathology & Laboratory Medicine,  Jul 1997  by Hinrichs, Steven H

2nd ed, by Gerald Nuovo, 449 pp, with illus, $80, New York, NY, Raven Press, 1994.

This book represents a renewed effort to bring procedures in molecular biology into the domain of anatomic pathology research. For pathologists who cut their teeth on avidin-biotin immunohistochemistry and only recently became comfortable with polymerase chain reaction (PCR) techniques, this book is a logical entry point into leading edge molecular diagnostics and research using PCR in situ hybridization...

http://findarticles.com/p/articles/mi_qa3725/is_199707/ai_n8764476

Nested in situ PCR-ISH method

Development of a novel nested in situ PCR-ISH method for detection of hepatitis C virus RNA in liver tissue

Authors: Alzahrani A.J.; Vallely P.J.; McMahon R.F.T.¹

Source: Journal of Virological Methods, Volume 99, Number 1, January 2002 , pp. 53-61(9)

Publisher: Elsevier

http://www.ingentaconnect.com/content/els/01660934/2002/00000099/00000001/art00383

Co-labeling Using In Situ PCR

Journal of Histochemistry and Cytochemistry, Vol. 49, 1329-1340, November 2001, Copyright © 2001, The Histochemical Society, Inc.

REVIEW

Co-labeling Using In Situ PCR: A Review

Gerard J. Nuovo^a
a Department of Pathology, Ohio State University Medical Center, Columbus, Ohio

Correspondence to: Gerard J. Nuovo, Dept. of Pathology, Ohio State U. Medical Center, S305 Rhodes Hall, 450 W 10th Ave., Columbus, OH 43210. E-mail: gnuovomd@pol.net

Summary

In situ amplification permits the histological localization of low-copy DNA and RNA targets. However, in many instances it would be useful to know the specific phenotype of the target-containing cell or to ascertain the distribution of a different nucleic acid sequence in the same tissue section. This review describes a methodology that allows co-in situ localization of two nucleic acid targets or a DNA/RNA sequence and a protein in paraffin-embedded, formalin-fixed tissue. The key variable for detection of low-copy RNA targets by RT in situ PCR is optimal protease digestion to permit cDNA target-specific incorporation of the reporter nucleotide. This is achieved via inactivation of nonspecific DNA synthesis by overnight DNase digestion. The key variable for immunohistochemical localization of proteins is to determine the effect of protease digestion on the antigen-based signal intensity. Background for DNA targets by in situ hybridization or, for targets present in 1–10 copies per cell, PCR ISH is dependent primarily on probe concentration and the stringency of the post-hybridization wash. Radioactive ³H-labeled nucleotides permit an excellent distinction with colorimetric signals for co-localization, although two distinct chromogens can in many instances allow successful localization of two different targets.

http://www.jhc.org/cgi/content/full/49/11/1329

In situ reverse trancriptase-polymerase chain reaction

J Nanobiotechnology. 2003; 1: 3.

Published online 2003 May 28. doi: 10.1186/1477-3155-1-3.

 

Copyright © 2003 Stamps et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

 

Application of in situ reverse trancriptase-polymerase chain reaction (RT-PCR) to tissue microarrays

Alasdair C Stamps,¹ Jonathan A Terrett,¹ and Paul J Adam¹

¹Oxford GlycoSciences (UK) Ltd., The Form, 86 Milton Park, Abingdon, UK OX14 4RY

Corresponding author.

Alasdair C Stamps: astamps@ntlworld.com ; Jonathan A Terrett: jon.terrett@ogs.co.uk ; Paul J Adam: paul.adam@ogs.co.uk

 

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=161785