Molecular Biology Services and DNA Sequencing Brochure

Description: Molecular Biology Services and DNA Sequencing DNA Sequencing Single Pass SequencingPurpose Sequence data only, for plasmids or PCR productsCustomer supplies• Plasmid DNA or PCR products Plasmid DNA: 20 – 100 ng/µl PCR Product: 5 – 50 ng/µl Volume: 20 µl or more (enough for 4 reactions) DNA dissolved in water• Custom primers 2 pmol/µl, minimum of 20 µlService includes• DNA quantif ication and dilution for optimum sequencing results• Storage of DNA samples and primers for future sequencing reactions for four weeks• Visual inspection of data before release to customer• One repetition of any failed sequencing reaction• Use of a special sequencing reaction mixture, where necessary, for dif f icult templates (GC-rich sequences, hairpin loops) • Technical support by specialistsCustomer receives• Notif ication by email that results are available• Results for download from a secure server for up to one month• Sequence data in .ab1 (chromatograms) and text format (.seq) • Read length up to 900 bases• Results within 24 hoursOptional Services• Plasmid Miniprep or PCR purif ication• Custom primer design or primer purchaseDNA Sequencing Pre-cycled Sequencing ReactionsPurpose A fast and economical service giving sequence data only, for plasmids or PCR productsCustomer supplies• Non purif ied sequencing reactions (Applied Biosystems Big Dye version 3.1) in tubes or in PCR plates• A minimum of 24 reactionsService includes• Purif ication of the sequencing reactions• Analysis of the purif ied sequencing reactions using an AB3730XL DNA Sequence Analyzer• Technical support by specialistsCustomer receives• Notif ication by email that results are available• Results for download from a secure server for up to one month• Sequence data in .ab1 (chromatograms) and text format (.seq) • Read length up to 900 bases• Results within 24 hoursDNA Sequencing Double-Stranded Sequencing for ConfirmationPurpose Complete sequence verif ication of a DNA sequence of interest on both DNA strands (raw data production, contig alignment and editing, comparison of consensus sequence to customer’s reference sequence)Customer supplies• Plasmid DNA or plasmid carrying E. coli culture (i.e., stab culture, glycerol stock, agar plates)• The sequence of the construct or region of interestService includes• Primer design and synthesis• Plasmid template preparation (free upon request)• DNA sequencing (double stranded sequencing)• Storage of DNA samples and primers for future sequencing reactions for four weeks• Sequence data alignment and editing of the resulting consensus• Technical support by specialistsCustomer receives• Notif ication by email that results are available• Results for download from a secure server for up to one month• A consensus sequence for the sequenced region(s)• A comparison of the consensus sequence to the provided reference sequence• A report listing all observed differences • Results within 24 hours (3-4 days if primers need to be ordered)DNA Sequencing Single-Stranded Sequencing for ConfirmationPurpose Complete verif ication of a DNA sequence of interest on one DNA strand only (raw data production, sequence alignment and editing, comparison of consensus sequence to customer’s reference sequence)Customer supplies• Plasmid DNA or plasmid carrying E. coli culture (i.e., stab culture, glycerol stock, agar plates)• The sequence of the construct or region of interestService includes• Primer design and synthesis• Plasmid template preparation (free upon request)• DNA sequencing (single stranded sequencing)• Storage of DNA samples and primers for future sequencing reactions for four weeks• Sequence data alignment and editing of the resulting consensus• Technical support by specialistsCustomer receives• Notif ication by email that results are available• Results for download from a secure server for up to one month• A consensus sequence for the sequenced region(s)• A comparison of the consensus sequence to the provided reference sequence• A report listing all observed differences • Results within 24 hours (3-4 days if primers need to be ordered)DNA Sequencing Primer Walking, Non-assembled Single Stranded SequencingPurpose The complete sequence of a DNA sequence of interest on one DNA strand (non assembled single reads)Customer supplies• Plasmid DNA or plasmid carrying E. coli culture (i.e., stab culture, glycerol stock, agar plates)• The sequence of the construct or region of interestService includes• Primer design and synthesis• Plasmid template preparation (free upon request)• Storage of DNA samples and primers for future sequencing reactions for four weeks• DNA sequencing• Visual inspection of raw data before release to customer• Technical support by specialistsCustomer receives• Notif ication by email that results are available• Results for download from a secure server for up to one month• Sequence data in .ab1 (chromatograms) and text format (.seq) • Read length up to 900 bases• Results within 24 hours (3-4 days if primers need to be ordered)DNA Sequencing Primer Walking, Non-assembled Double Stranded SequencingPurpose The complete sequence of a DNA sequence of interest on both DNA strands (non assembled single reads)Customer supplies• Plasmid DNA or plasmid carrying E. coli culture (i.e., stab culture, glycerol stock, agar plates)• The sequence of the construct or region of interestService includes• Primer design and synthesis• Plasmid template preparation (free upon request)• Storage of DNA samples and primers for future sequencing reactions for four weeks• DNA sequencing• Visual inspection of raw data before release to customer• Technical support by specialistsCustomer receives• Notif ication by email that results are available• Results for download from a secure server for up to one month• Sequence data in .ab1 (chromatograms) and text format (.seq) • Read length up to 900 bases• Results within 24 hours (3-4 days if primers need to be ordered)DNA Sequencing cGMP SequencingPurpose Confirmation of a DNA sequence under cGMPDescriptionDNA sequencing under cGMP is based upon a generically validated method. Analysis under cGMP is preceded by product specif ic verif ication.Verification of product specific parameters for cGMP sequencingProduct-specif ic sequencing conditions are determined via a non-cGMP(ISO9001) study. The conditions are summarized in a QA approved parameter sheet that includes: • Primer design and primer synthesis• Double stranded DNA sequencing (at least 2 fold strand coverage)• Development of product-specif ic sequencing conditions• Description of product-specif ic sequencing conditions in a parameter sheetcGMP sequencingPerformed under cGMP using the product-specif ic sequencing conditions to include: • The usage of qualif ied equipment• cGMP trained and qualif ied personal• Documentation complying with cGMP guidelines• Double stranded DNA sequencing with at least 2 fold strand coverageIf the specif ic sequencing conditions elaborated during verif ication differ from the validated range, then validation of the specif ic conditions will be performed. Customer supplies• Plasmid DNA: 20 - 1000 ng/µl (at least 5 µg/kb). • DNA dissolved in water.• The reference sequence of the construct or region of interest.• The specif ication of the product.Customer receives• The analytical results under cGMP• The consensus sequence for the sequenced region• A sequence alignment with a comparison of the consensus sequence to the reference sequence• A summary of the DNA sequencing method together with a summary of the verif ication of product specif ic parameters• The parameter sheet• Results within 4 -6 weeks after receipt of samplesOptional Services• Plasmid Miniprep or Maxiprep (under ISO9001)• A full cGMP sequencing report : summary of DNA sequencing strategy, experiments and results; all primer sequences; all sequencing reactions; the sequence alignment; the consensus sequence and the reference sequence.DNA Sequencing Exon SequencingPurpose Sequence verif ication of a genomic region of interest in several individuals (i.e., cell lines, diagnostic families members) and comparison to a reference sequence (i.e., SNP analysis)Customer supplies• Sequence information concerning the gene or genomic region of interest• Genomic DNA of individuals to be analyzed Service includes• PCR amplif ication of region(s) of interest (maximum 600 nt)• Sequence analysis of resulting PCR product• Comparison of amplicon sequence to customer’s reference sequenceCustomer receives• Notif ication by email that results are available• Results for download from a secure server for up to one month• Detailed report of the SNP analysisPrimer for DNA Sequencing Custom primerPurpose For sequencing onlyCustomer supplies• Primer sequenceService includes• Primer synthesisPrimer for DNA Sequencing Primer designPurpose For sequencing onlyCustomer supplies• Reference sequenceService includes• Primer design• Primer synthesisDNA Preparations Plasmid MiniprepPurpose For sequencing onlyCustomer supplies• Plasmid carrying E. coli culture (i.e., stab culture, glycerol stock, agar plates)Service includes• Purif ication of the plasmidDNA Preparations PCR Product PurificationPurpose For sequencing onlyCustomer supplies• A non purif ied PCR productService includes• Purif ication of PCR product using QIAGEN methodDNA Preparations Large-Scale, Endotoxin-free Plasmid PrepPurpose An endotoxin-free plasmid maxi-prep (100 microgram scale)Customer supplies• Plasmid carrying E. coli culture (i.e., stab culture, glycerol stock, agar plates) or plasmid DNAService includes• Purif ication of the plasmid• Photometric quantif ication of resulting DNA• Identity test by DNA sequencingCustomer receives• Endotoxin-free plasmid DNA• Minimum of 100 µg of DNA (turnaround time 1 week)• Sequence data and reportOptional Services• Complete double or single stranded confirmation sequencing. Transformation E. coli TransformationPurpose For sequencing onlyCustomer supplies• Plasmid DNAService includes• E. coli transformationOptional Services• Plasmid MiniprepSite-Directed Mutagenesis Site-Directed Mutagenesis (Single Site)Purpose To introduce a change into a sequence of interest (single locus change).To verify the sequence of the mutated site.Customer supplies• Plasmid DNA• Reference sequenceService includes• Site directed mutagenesis of site of interest• Sequence verif ication of the mutated site(s) (typically 500 nt around the mutated site will be verif ied).Customer receives• Mutated construct (turnaround time 1-2 weeks)• Sequence data and reportOptional Services• Complete double or single stranded confirmation sequencing of mutagenised clone (price according to list). • Endo-toxin free plasmid maxiprepSite-Directed Mutagenesis Site Directed Mutagenesis (Multiple Sites)Purpose To introduce multiple changes into a sequence of interest (multiple loci change)To verify the sequence of the mutated sites.Customer supplies• Plasmid DNA• Reference sequenceService includes• Site directed mutagenesis of sites of interest• Sequence verif ication of the mutated sites (typically 500 nt around the mutated sites will be verif ied)Customer receives• Mutated construct (turnaround time 1-2 weeks)• Sequence data and reportOptional Services• Complete double or single stranded confirmation sequencing of mutagenised plasmid (price according to list)• Endo-toxin free plasmid maxi-prepClone Screening by SequencingPurpose • A positive clone screened out of multiple clones. • Sequence verif ication at the site of interest of the positive plasmid• DNA preparation of plasmid templateCustomer supplies• Plasmid DNA of clones to be screened (4 minimum)• Plasmid-carrying E. coli culture (i.e., stab culture, glycerol stock, agar plates) of clones to be screened (4 minimum)• The reference sequenceService includes• DNA preparation of plasmid templates• Sequencing of each clone with 2 primers of choice (primer design and purchase included• Alignment to the reference sequencesCustomer receives• Sequence data• Report on positive clones identif iedOptional Services• Complete double or single stranded confirmation sequencing of one (or more) positive clones (price according to list)• Endotoxin free plasmid Maxiprep of a positive cloneSubcloning (via PCR or Restriction Enzyme Digestion)Purpose Subcloning of a DNA fragment (PCR product or restriction digestion) into a vector of choiceCustomer supplies• Plasmid DNA• Plasmid-carrying E. coli culture (i.e., stab culture, glycerol stock, agar plates)• The reference sequencesService includes• Subcloning of a DNA fragment (PCR product or restriction digestion) into a vector of choice• Screening for a positive clone• 5 and 3 prime end sequencing and alignment to the reference sequenceCustomer receives• Sequence data• Report on positive clones identif ied• Plasmid DNA of positive cloneOptional Services• Complete double or single stranded confirmation sequencing of one (or more) positive clones (price according to list)• Endo-toxin free plasmid Maxiprep of a positive cloneCloning of cDNA or Genomic DNAPurpose Cloning of a DNA fragment or a gene of interest into a vector of choiceCustomer supplies• DNA sequence informationService includes• Cloning in a vector of choice of the DNA sequence of interest• Complete sequence verif ication of the isolated clonesCustomer receives• Plasmid DNA of isolated clone• Sequence dataOptional Services• Endo-toxin free plasmid Maxiprep of a positive clone Cloning of Short Hairpin RNAs (shRNAs)Purpose Cloning of short hairpin RNA into a vector of choiceCustomer supplies• The reference sequence of the vector• The shRNA sequencesService includes• Purchase of sh-Oligos • Cloning of sh-Oligos into a vector of choice• Screening for a positive clone• 5 and 3 prime end sequencing and alignment to the reference sequenceCustomer receives• Sequence data • Report on positive clones identif ied • Plasmid DNA of positive cloneOptional Services• Endotoxin-free plasmid Maxiprep of a positive clone