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

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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

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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

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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

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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 -