Sanger Sequencing Services Order Preparation and Submission

DNA Sequencing Quick Guide before Submitting the Order

Please ensure that your samples meet our requirements.

Download Sample Preparation Quick Guide below to aid the speedy preparation of your samples.

Singapore CustomersMalaysia CustomersInternational Customers
DNA Sequencing Quick GuideDNA Sequencing Quick GuideDNA Sequencing Quick Guide

Single Pass DNA Sequencing Sample Requirements

For Singapore & Malaysia Customers

Type of DNA Template

Recommended Concentration/ Reaction

Required Volume/ Reaction

Purified PCR Product [90-500bp]

5 ng/µL

10 uL

Purified PCR Product [501-1500bp]

20 ng/µL

Purified PCR Product [>1500bp]

30 ng/µL

Purified Plasmid (< 4kb)

50 ng/µL

Purified Plasmid (4kb to 10kb)

80 ng/µL

Purified Plasmid (>10kb)

100 ng/µL

Purified M13/ phagemid ssDNA

50 ng/µL

Purified Genomic DNA/ BAC

400 ng/µL

20 uL

 

Type of Primer

Required Concentration

Total of Reaction

Required Volume

Sequencing Primer

  • 18 to 25 bases
  • GC content: 40% to 60%
  • Tm = 55°C to 60°C
  • No significant hairpins (>3bp) and homodimerization
  • Desalted Primer (Brand: IDT) or Purified Primer (other Manufacturer)

10µM or 10pmol/µL or 50ng/uL

1

10uL

10

25uL

20

50uL

 

For International Customers

We recommend that international customers send a larger volume of their samples to overcome loss of samples due to evaporation during the extended transportation to our facilities.

Type of DNA Template

Recommended Concentration/ Reaction

Required Volume/ Reaction

Purified PCR Product [90-500bp]

5 ng/µL

15 uL

Purified PCR Product [501-1500bp]

20 ng/µL

Purified PCR Product [>1500bp]

30 ng/µL

Purified Plasmid (< 4kb)

50 ng/µL

Purified Plasmid (4kb to 10kb)

80 ng/µL

Purified Plasmid (>10kb)

100 ng/µL

Purified M13/ phagemid ssDNA

50 ng/µL

Purified Genomic DNA/ BAC

400 ng/µL

25 uL

 

Type of Primer

Required Concentration

Total of Reaction

Required Volume

Sequencing Primer

  • 18 to 25 bases
  • GC content: 40% to 60%
  • Tm = 55°C to 60°C
  • No significant hairpins (>3bp) and homodimerization
  • Desalted Primer (Brand: IDT) or Purified Primer (other Manufacturer)

10µM or 10pmol/µL or 50ng/uL

1

10uL

10

25uL

20

50uL

 

Sequencing+ PLUS Sample Requirements

For Unpurified Samples

 

Sample Type

General Guideline

Unpurified PCR Products

20µL

 in 1.5ml tube/ plate

Excised Gel Band

<250mg

 in 1.5mL tube

For Plasmid (Singapore Customers)

Click here to enlarge image.

Direct Colony Sequencing Sample Requirements

Sample Type

 Colony Size

Slant surface agar

Pick a single colony using sterile inoculating loop and streak on the surface of the prepared agar.

Incubate the culture at optimum growing temperature for overnight. Ensure that there is visible growth of the bacteria colony to the indicated size in Figure below:

Size of the colony should be approximately 1mm (width) X 6 mm (length).

 

NOTE: Do not stab your culture into the agar. The colony should grow on the surface of the agar for better visibility of the colony size.

 

Straight surface agar

M13-Tailed Sequencing Sample Requirements

For Malaysia & International Customers

 

Sample Type

Purification

Specification

Size and Volume

Disclaimers

qPCR Products

Only unpurified qPCR/ PCR products are accepted

Ct value < 33

> 90 bp, minimum 15µL

(i) M13-tailed amplification needs ~ 5ng of DNA amount for each reaction.
(ii) Failed amplification will be charged for proviso fee.

PCR Products

Visible on agarose gel using 2µL

> 90 bp, minimum 15µL

Primer Walking Sample Requirements

Length

Concentration & Volume/ Project

Primer Walking [<= 3kb]

100 ng/µL, min 50 µL per rxn in dH2O

1.5kb per week

Primer Walking [> 3kb]

Whole bacterial colony OR 100 ng/µL, min 50 µL per rxn in dH2O

 

 

Primer Walking Sample Requirements

Length

Concentration & Volume/ Project

Primer Walking [<= 3kb]

100 ng/µL, min 50 µL per rxn in dH2O

1.5kb per week

Primer Walking [> 3kb]

Whole bacterial colony OR 100 ng/µL, min 50 µL per rxn in dH2O

Sample Preparation Requirements for DNA Purification & Quantification Services

We recommend that international customers send a larger volume of their samples to overcome loss of samples due to evaporation during the extended transportation to our facilities.  

Sample Preparation and Quantification Service

Malaysia & Singapore

International

Required Concentration & Volume

Quantification by UV Spectrometry (SS1021)

min. 15 µL for each sample

min. 25 µL for each sample

Quantification by Agarose Gel (SS1020)

min. 15 µL for each sample

min. 25 µL for each sample

Purification Using PCR Clean-up (SS1012/3)

100 ng/µL, min 20 µL per reaction in dH2O

100 ng/µL, min 40 µL per reaction in dH2O

Purification Using Gel Extraction (SS1014/5)

100 ng/µL, min 30 µL per reaction in dH2O

100 ng/µL, min 40 µL per reaction in dH2O

Ready-to-Load Sequencing with Clean-up (SS1019)

min. 10 µL, please indicate cycle reaction volume.

min. 20 µL, please indicate cycle reaction volume.

Packing of Samples (DNA Templates and Sequencing Primers)

  1. Please prepare Sample in microcentrifuge tube (preferably in 1.5mL tubes) or directly on 96-well plate for high-throughput sequencing orders (empty the well no. H12 for our internal control reaction)
  2. Label clearly on the side and top of tube with a permanent marker
  3. Please use simple labels on your tubes (e.g. initials and numbers) and avoid long sample names
  4. Ensure that the lid is tightly closed and seal the tubes with parafilm
  5. Place samples in a plastic bag and attach the completed DNA Sequencing order form

Guidelines for packaging for safe transportation of DNA sequencing samples from Outstationed / International Customers:

  1. Please prepare Samples in microcentrifuge tube (preferably in 1.5mL tubes) or directly on 96-well plate sealed with strip-caps for high-throughput sequencing orders (empty the well no. H12 for our internal control reaction)
  2. Label clearly on the side and top of tube with a permanent marker
  3. Please use simple labels on your tubes (e.g. initials and numbers) and avoid long sample names
  4. Ensure that the lid is tightly closed and seal the lid with parafilm
  5. Place samples in a plastic bag. Do not stick samples directly onto order forms, please attach order form to the plastic bag instead.
  6. Wrap the plastic bag with a bubble bag and send in a padded envelope by overnight delivery either through Courier. DNA samples can be sent at room temperature.
  7. Notify 1st BASE via email and provide tracking number if any.

When in doubt, do not hesitate to contact us for any clarifications.

How to Pack and Seal 96-Well for Malaysia & Singapore Customers

How to Pack and Seal 96-Well for International Customers