Show simple item record

dc.contributor.authorKim, Tae Hoon
dc.contributor.authorDekker, Job
dc.date2022-08-11T08:10:59.000
dc.date.accessioned2022-08-23T17:27:27Z
dc.date.available2022-08-23T17:27:27Z
dc.date.issued2018-08-01
dc.date.submitted2018-09-19
dc.identifier.citation<p>Cold Spring Harb Protoc. 2018 Aug 1;2018(8):pdb.prot097865. doi: 10.1101/pdb.prot097865. <a href="https://doi.org/10.1101/pdb.prot097865">Link to article on publisher's site</a></p>
dc.identifier.issn1559-6095 (Linking)
dc.identifier.doi10.1101/pdb.prot097865
dc.identifier.pmid30068592
dc.identifier.urihttp://hdl.handle.net/20.500.14038/49864
dc.description.abstractControl libraries are used to determine differences in the efficiency of polymerase chain reaction (PCR) primers in detecting 3C ligation products as well as any differences in the efficiency of amplification. Control libraries are generated by random ligation of restriction fragments so that each ligation product is present in equimolar amounts. This protocol begins with preparations of genomic DNA or BAC DNA preparations. The restriction enzyme used here should be the same as the one used for generation of the 3C or ChIP-loop library. For small genomes, such as those of bacteria and yeast, this protocol can be started with purified genomic DNA. For organisms with larger genomes, such as mouse and human, control libraries are generated from purified BAC clones that cover the genomic region under study. When the region of interest is large and multiple BAC clones are required, select BAC clones that overlap only minimally. Any gaps between the regions covered by the BACs should also be kept to a minimum. Several kits for purifying BAC clones are available (e.g., the Large-Construct Kit from QIAGEN). BAC clones should be mixed in equimolar amounts to ensure that each restriction fragment is present in equal concentration. This is achieved by quantifying the molar concentration of each BAC preparation using real-time PCR and primers that recognize the common BAC vector backbone. Alternatively, the concentration of BAC preparations can be quantified using gel quantification or a NanoDrop detection system.
dc.language.isoen_US
dc.relation<p><a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=30068592&dopt=Abstract">Link to Article in PubMed</a></p>
dc.relation.urlhttps://doi.org/10.1101/pdb.prot097865
dc.subjectBiochemistry, Biophysics, and Structural Biology
dc.subjectGenetics and Genomics
dc.subjectLaboratory and Basic Science Research
dc.subjectSystems Biology
dc.titleGeneration of Control Ligation Product Libraries for 3C Analyses
dc.typeJournal Article
dc.source.journaltitleCold Spring Harbor protocols
dc.source.volume2018
dc.source.issue8
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/sysbio_pubs/136
dc.identifier.contextkey12900763
html.description.abstract<p>Control libraries are used to determine differences in the efficiency of polymerase chain reaction (PCR) primers in detecting 3C ligation products as well as any differences in the efficiency of amplification. Control libraries are generated by random ligation of restriction fragments so that each ligation product is present in equimolar amounts. This protocol begins with preparations of genomic DNA or BAC DNA preparations. The restriction enzyme used here should be the same as the one used for generation of the 3C or ChIP-loop library. For small genomes, such as those of bacteria and yeast, this protocol can be started with purified genomic DNA. For organisms with larger genomes, such as mouse and human, control libraries are generated from purified BAC clones that cover the genomic region under study. When the region of interest is large and multiple BAC clones are required, select BAC clones that overlap only minimally. Any gaps between the regions covered by the BACs should also be kept to a minimum. Several kits for purifying BAC clones are available (e.g., the Large-Construct Kit from QIAGEN). BAC clones should be mixed in equimolar amounts to ensure that each restriction fragment is present in equal concentration. This is achieved by quantifying the molar concentration of each BAC preparation using real-time PCR and primers that recognize the common BAC vector backbone. Alternatively, the concentration of BAC preparations can be quantified using gel quantification or a NanoDrop detection system.</p>
dc.identifier.submissionpathsysbio_pubs/136
dc.contributor.departmentDepartment of Biochemistry and Molecular Pharmacology
dc.contributor.departmentProgram in Systems Biology
dc.source.pagespdb.prot097865


This item appears in the following Collection(s)

Show simple item record