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Submission Guide


Tube & Plate Sequencing

SimpleSeq

Additional Services & Support

- Volumes and Concentrations

- Prepared Template and Primer

- Separate Template and Primer

- Pre-mixed Template & Primer

- Preparation / Purification

- Shipping Address & Information

- SimpleSeq & Tube Submissions

- Plate Sequencing

- Standard Plates

- Express Plates

- RTL Xpress Plates

- SimpleSeq

- SimpleSeq: Webless

- Order SimpleSeq

- Activate SimpleSeq Kits

- Free Overnight Shipping Kits

- UPS Drop-off Sites

- Service Types

- Improving DNA Sequencing Results

- Power Read Service

- RCA Plasmid Preparation

- Primer Selection

- Standard Primer List

- Price List

Submitting Templates & Primer Samples: Concentrations & Volumes

I. Concentrations

Table 1: Prepared Template and Primer Submissions

Template Type

Template Size

[Template] (ng/uL)

[Primer]

(pmols/uL)

Plasmid

<20 Kb

100-200

2-10

Purified PCR

100-300 bp

10-20

2-10

Purified PCR

301-1000 bp

20-40

2-10

Purified PCR

>1000 bp

40-60

2-10

Large Constructs (BAC)

>20 kb

250-500

10-15


II.Volumes

Separate Template and Primer

  • Your template DNA is being submitted separately from your primer samples.
  • Sample and primer will be mixed by our technicians prior to sequencing.

Table 2: Volume Required for Separate Template and Primer Submissions*

Single Aliquot

Volume (1st Rxn)

Additional Volume (Each Additional Rxn)

Template DNA

10 uL

5 uL

Primer

10 uL

5 uL

Power Read DNA

10 uL

10 uL

*Please see Table 1 for relevant concentrations

Pre-mixed Template and Primer

  • Your template DNA is being submitted already mixed with your primer (1 primer per tube).
  • Adhere to concentrations set-forth in Table 1 for stock solutions prior to mixing.

Table 3: Pre-mixed Sample Submissions

Template Volume (uL)

Primer Volume (uL)

Total Volume (uL)

8

4

12


Samples for Preparation / Purification

· Your template DNA requires further preparation or purification prior to sequencing.

  • Primer should always be submitted separate from sample in this instance.
  • See Table 3 for further volume and primer specifications.

Template Type

[Primer] (pmols/uL)

Submission Volumes (uL)

Crude PCR For Purification (>100 bp)

2-10

Entire Reaction Volume (15-30)

Bacterial Cultures for RCA Plasmid Preparation

2-10

10 uL Glycerol Stocks Recommended


Shipping Address & Information

How to Ship Samples

Enclose your tubes in our Blue Bag and ship in a padded envelope to the following address:

Eurofins Genomics

DNA Sequencing Services
1044 E Chestnut St
Louisville, KY 40204-6033

USA

If you have submitted an online order, please enclose a copy of the online order confirmation page with every shipment to ensure proper identification, tracking, and result retrieval.


Free Overnight Shipping Kits Available!

Your FREE Overnight Kit will contain:

- 8 UPS shipping envelopes personalized for your lab

- 16 bar-coded labels for unique template and primer ID

- 8 sequencing sample bags

New! SimpleSeq™ Webless™ Pre-Mixed Reaction Submissions

  1. Prepare aqueous template and primer solutions at the concentrations listed in Table 2.
  2. Add 8 μL template + 4 μL primer to each tube by inserting your pipet tip through the split seal (total volume =12 μL). See Table 2 for more information.

SimpleSeq and Tube Submissions

  1. Prepare aqueous template and primer solutions at the concentrations listed above
  2. Place a barcoded label on any tubes without a barcode. SimpleSeq tubes will be pre-labeled.
  3. Aliquot templates and primers as follows (per Table1)
  4. Free standard primers can be added by our lab
  5. Enter the details of your reactions in our online order form

Plate Submission

(Standard, Express, Ready to Load)

  1. Samples should be shipped using 96-well, v-bottom, full-skirt PCR plates
  2. Two wells on each plate should be left empty for our internal quality controls at positions H11/H12 for row layouts and G12/H12 for column layouts
  3. Enter the detail for your reactions in our online order form
  4. Only strip caps provide the most secure shipping conditions (we do not recommend foil or tape-seals as cross-contamination and sample loss can frequently occur)
  5. Please attach one barcode label to each plate for unique identification

Standard Plate Sequencing

- Full length analysis protocol

- Minimum of 48 reactions per plate

- Online order required

Express Plate™ Sequencing

- Prepare aqueous template and primer solutions at the concentrations listed above in Table 2

- Using 96-well, v-bottom, full-skirt PCR plates, add 8 μL template + 4 μL primer to each well (total volume =12 μL)

- Note that Express plates are processed using an abbreviated analysis protocol (for improved turnaround time) and read lengths are 500-700 bp

Ready to Load Plate Sequencing

- Only full-skirted, ready to load plates are accepted for analysis

- Please make sure your BigDye v3.1 reaction arrives post-purification and the order has been placed

- Note that RTL plates are processed using an abbreviated analysis protocol (for improved turnaround time) and read lengths are 500-700 bp

- Dried samples are preferred

Power Read Service*

Utilizes a proprietary chemistry, geared towards resolving sequence across difficult stretches of template DNA

Ideal for Sequencing the Following:

GC-rich templates

Difficult secondary structures

Homopolymeric sequences

Repetitive Sequence

Bisulfite-treated DNA

shRNA

Includes one automatic repeat read for reactions which fail or generate insufficient sequence quality on the initial run

(*Repeat reads may be may not be possible if samples are submitted outside the recommended volume conditions)

RCA Plasmid Preparation Service

  • Glycerol stocks are highly recommended
  • Send a minimum of 10 μL for each sample (50% glycerol solution)
  • Prepare stocks from overnight cultures
  • Glycerol stocks, pelleted cells, overnight cultures, and agar plates are all accepted submission methods and all should be shipped overnight on cold packs

Primer Selection

Free standard primers

  • Over 60 standard vector primers are available for sequencing
  • See our Primer Library for a comprehensive list

Primer Design & Storage

  • 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
  • Primer walking, primer design, and primer synthesis services are also available

For Best Results:

  • Suspend templates in water only as other buffers may inhibit the sequencing reaction (specifically excessive concentrations of EDTA)
  • Sequencing templates should be provided as purified plasmid, BAC, or PCR product (RCA Plasmid prep and PCR purification by requesting “Template Prep” within your order submission – BAC preparation services are available by e-mail order form)

- Need help troubleshooting your sequencing data? Feel free to contact our Technical Support team at 1-800-688-2248 (option2), e-mail us at dnatechsupport@eurofins.com, or review our Troubleshooting Guide online.

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Eurofins MWG Operon, Inc. A Eurofins Genomics company. All rights reserved.
Eurofins Genomics | 2211 Seminole Drive | Huntsville, Alabama 35805, USA
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