DNA Sequencing Sample Submission Guidelines

For rapid processing of your automated DNA sequencing samples and to obtain the best results, please follow these template and primer submission guidelines.

All Samples and Services

Sequencing templates should be provided as purified plasmid, BAC, or PCR product
Plasmid prep, PCR purification, and BAC culturing services are available, if requested
Suspend templates in water only; other buffers may inhibit the sequencing reaction

Recommended Concentrations & Volumes
Template	Concentration	Primer Sequence
Plasmid	100–200 ng/µl	10 µl for 1st reaction + 5 μL per add’l reaction
PCR <300 Bp	10–20 ng/µl	10 µl for 1st reaction + 5 μL per add’l reaction
PCR 300–1000 Bp	20–40 ng/µl	10 µl for 1st reaction + 5 μL per add’l reaction
PCR >1000 Bp	30–60 ng/µl	10 µl for 1st reaction + 5 μL per add’l reaction
BAC (>20) kBp	500–1000 ng/μL	10 µl for 1st reaction + 5 μL per add’l reaction
Primer (plasmid or PCR)	2 μM (pmol/μL)	10 µl for 1st reaction + 5 μL per add’l reaction
Primer (BAC)	10 μM (pmol/μL)	10 µl for 1st reaction + 5 μL per add’l reaction

Please review your DNA sequencing order summary sheet for exact recommendations based on your specific order.

Templates and Primers in Tubes

Provide template and primer at recommended concentrations (see table above) based on template type
Samples should be shipped using either our SimpleSeq™ tubes or 1.5 – 2.0 mL microfuge tubes labeled with the free barcodes

Templates and Primers in Plates

Provide template and primer at recommended concentrations (see table above) based on template type
Samples should be shipped in 96-well, v-bottom, full-skirt PCR plates (min. 48 reactions per plate)
Please attach one barcode label to each plate for unique identification
Two wells on each plate should be left empty for our internal quality controls at positions H11/H12 for row layouts or G12/H12 for column layouts
Only strip caps or cap mats should be used for sealing (we do not recommend foil or tape-seals as cross-contamination and sample loss can frequently occur)

Premixed Samples with Primers

Template and primer solutions should follow the concentrations listed in the above table prior to mixing
Mix 8 μL of template and 4 μL of primer for a total volume of 12 μL for each reaction

Express Plate™ DNA Sequencing Services

Use capped, 96-well, v-bottom, full-skirt PCR plates
Follow sample submission guidelines specified above for pre-mixed samples
Please attach one bar-coded label to each plate for unique identification

Express Plate Ready to Load Service

Only reactions using ABI BigDye Terminator Kit (v3.1 or v1.1) can be accepted
The dye terminator clean-up procedure must be performed before submitting samples
Dried samples are preferred

Primer Selection

Many standard vector primers are available for sequencing (see partial list below)
Primer walking, primer design, and primer synthesis services are also available
Primers synthesized or shipped to us for sequencing should be between 18 and 22 bp in length, avoid stretches of
identical nucleotides, display about 50% GC content, and exhibit an annealing temperature of 50 – 60°C
Primers that are synthesized for your sequencing order remain available to use for six months

Partial List of Standard Primers
Vector Primer	Primer Sequence	Vector Primer	Min. Volumes
M13F	TGT AAA ACG ACG GCC AGT	T7Term	CTA GTT ATT GCT CAG CGG T
M13R	CAG GAA ACA GCT ATG ACC	pBadF	ATG CCA TAG CAT TTT TAT CC
M13F43	AGG GTT TTC CCA GTC ACG ACG TT	pBadR	GAT TTA ATC TGT ATC AGG
M13R49	GAG CGG ATA ACA ATT TCA CAC AGG	petup	ATG CGT CCG GCG TAG A
SP6	CA TTT AGG TGA CAC TAT AG	pGexF	ATA GCA TGG CCT TTG CAG G
T3	AAT TAA CCC TCA CTA AAG GG	pGexR	GAG CTG CAT GTG TCA GAG G
T7	TAA TAC GAC TCA CTA TAG GG	BGHrev	TAG AAG GCA CAG TCG AGG

Plasmid Preparation Service

When providing samples as glycerol stocks or pelleted cells, ship overnight on dry ice
When provinding samples as overnight cultures or isolated colonies, ship overnight with ice packs

Sample Submission for PCR Purification

Dispense 10 – 50 μL of PCR product in 1.5 – 2.0 mL microfuge tubes or 96-well, full-skirt PCR plates
Seal plates only with strip caps or a cap mat (avoid foil or tape-seals)

Ordering DNA Sequencing Services

Visit www.operon.com and click the "Order Now" button on the right of your screen
Select "Order – DNA Sequencing" and follow instructions for Tube Entry, Tube Upload, Plate Entry, or
Plate Upload ordering

Ordering Free Bar-coded Labels, SimpleSeq Kits, and Free Sample Shipment Kits

Sample Shipment

Unless otherwise indicated, please ship templates and primers at room temperature via overnight delivery to the address specified on your order confirmation.

IMPORTANT: Please enclose a copy of the online order confirmation page with every shipment to ensure proper identification, tracking, and result retrieval. Using barcodes for all sample tubes and/or plates will serve as unique identification and allow fast, accurate, and secure retrieval of your results. However, enclosing the order confirmation page with your samples is still recommended

If you have questions about our DNA sequencing sample submission guidelines or the online order entry system, please call us between 7:00 am to 7:00 pm Central Time for immediate assistance or send an e-mail to dnatechsupport@eurofins.com

Sincerely,

Your Eurofins MWG Operon Support Team

Phone: 1 (800) 688-2248
Fax: 1 (251) 252-7794

Eurofins MWG Operon > Services > DNA Sequencing > Sample Submission Guidelines