The Sanger Sequencing - Quick Guide

Ready2 Run Flexi Run Premium Run 1-shot reaction n-reactions n-reactions

• Turnaround time 24 hours • Turnaround time 24 hours • Turnaround time ~ 24 hours (except for clones) • One run per sample/plate • Several runs per sample/plate • DNA quality check • Template/primer storage for three months • Protocol modifications possible • Several runs per sample/plate Purified template includingprimer Additional service: Primer synthesis concentration preadjusted • Template/primer storage for three months Additional services: Primer design and synthesis Purified template concentration preadjusted

Unpurifiedor purified Unpurifiedtemplate

template Clone plate or PCR plate

Total volume = 14 µl per Min. volume = or well 14 µl per tube / well (+4 µl per each add. run) Single sample MTP

Template/primer preadjustments mandatory Primers in tubes or mirror plate Purification at Plasmid prep and PCR purification Primer conc. for Ready2 Run: 4 µl of a 5 µM solution extra costs price included Template conc. Ready2 Run and Flexi Run: Plasmid (up to 12 kb): 100 ng/µl PCR: 150-500 bp: 10 ng/µl 500-1000 bp: 20 ng/µl Universal Primers in tubes or mirror plate 1000-2000 bp: 40 ng/µl barcode for all Sanger services

For scientific advice and customer support, please contact us at: Tel: +49 (0)30 5304 22 30/40 [email protected] or visit www.lgcgroup.com/genomics Online ordering system Custom primer requirements Shipping specification To place your order please visit our • Length of 18 - 25 bases for Single Sample Sequencing: Online shop and log in at • No wobble bases • Use 1.5 ml tubes preferably ‚safe lock‘ (no ) for https://shop.lgcgenomics.com • GC-content of at least 40% sample submission.

• Register as a new user. • The annealing temperature must be Tm-3 and should be at least 52°C. • Affix the appropriate to each tube. Send sufficient volume of

• Choose your sequencing service, order labels • To calculate Tm please use Tm=4x(G+C)+2x(A+T). template DNA - see preadjustments (page 1). (and shipping for MTP sequencing), • 3’- end should be G or C • All sample tubes must be correctly barcode labelled. order free sample , manage your data • No self-hybridisation (primer dimer, loops) or binding to several and shipment. sites on the template for MTP Sequencing: • 20 pmol per reaction or stock solution (100 µM) dissolved • Please prepare your samples according to • Use semi-skirted or skirted plates only. in ultrapure water. Please specify the concentration on the tube. the given requirements and send your • For PCR products: v-shaped plates • Deprotected samples in a padded (sample ). For clones: u-shape or flat bottom plates • Without modifications (fluorophore or others) • Please use 8-strip caps to seal the plates (preferably from the • Free of salts and other contaminants. same supplier). Film or foil sealing of plates is not sufficiently secure for shipping. Template DNA requirements for Single Sample Sequencing: • Since an equal volume of sample will be used for all sequencing The main factors influencing read length are the • Primer stocks should be submitted in 1.5 ml ‚safe-lock‘ tubes and reactions, please ensure that the fragments do not vary in size by DNA concentration and the DNA quality. individually labelled with primer name and concentration. more than a factor of three (i.e. the largest fragment is not more • Template DNA must be free of EtOH, EDTA, than three times the size of the smallest). Plasmid DNA should be RNA, salts, genomic DNA and proteins. for MTP Sequencing: at a uniform concentration across the plate. • Please use ultrapure water for elution. • We accept a maximum of 6 different primers per sample - 1 primer via courier service via post ( only) • Plasmid DNA should be present in covalently per tube. Templates for given primers have to be arranged in LGC Genomics GmbH LGC Genomics GmbH closed circular form. blocks (i.e. lines or rows). Ostendstr. 25 / TGS Haus 8 P.O. 940327 • PCR products need to appear as a single band • Where more than 6 primers are required for a single sample 12459 Berlin 12443 Berlin in an agarose gel and have to be purified from plate, or a block arrangement of templates is not possible, LGC Germany Germany reaction buffer, primers and nucleotides. recommends a ‚mirror plate‘ of primers . • LGC recommends silica membrane-based spin • The mirror plate should comprise 15 µl primer solution per well column kits for template purification. (5 µM or 5 pmol/µl). Sequencing results • Please use 8-strip caps to securely seal the primer mirror plate. Automated and standardised ABI 3730 XL sequencing run with a read length up to 1,100 bp (PHRED20 quality). General information Free universal primers and free mass gel standard available. All sequencing data are available for three months from the Sequencing of PCR fragments smaller than password-protected download area of the sequencing online 150 bp is not recommended due to base calling ordering system. limitations of the current technology. For scientific advice and customer support, We provide the chromatograms (.ab1 and .scf files) and the text For an accurate determination of the DNA please contact us at: sequence (.txt file) extracted by the LGC software. concentration of your plasmid please use the Tel: +49 (0)30 5304 22 30/40 LGC plasmid DNA standard which is supplied Additionally, the secure download area provides download links to [email protected] free of charge. free programmes for sequence data visualisation. or visit www.lgcgroup.com/genomics

www.lgcgroup.com/genomics • [email protected] Science for a safer world Brazil • Bulgaria • China • France • Germany • Hungary • India • Ireland • Italy • Netherlands Nordic countries • Poland • Romania • Russia • South Africa • Spain • Turkey • United Kingdom • USA

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