Difficult Template sequencing (SS1006)

This service caters specifically to templates with sequence context difficulties. Sequence context difficulties include high G%, high C%, high GC%, repeat regions and loop structures found in shRNA vectors. 1st BASE adopts a robust protocol that maximizes the success rate of sequencing through these regions. However, the success rates of GT and GC repeats have shown to be limited by this chemistry. Currently, there is no guaranteed to overcome such homopolymeric regions. Please refer our troubleshooting guide for more details.

Specifications

As compared to our regular sequencing services, a greater volume for each sample is to be submitted (in case a repeat run is required).

Sample Preparation Requirements for DNA Sequencing Reactions for local customers (Singapore and Malaysia)

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

 

Sample Preparation Requirements 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