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.
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 fragme1. 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
| 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 |
| 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
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
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
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]
