Products & ServicesApplications

Applications

Nanopore sequencing offers advantages in all areas of research. Our offering includes DNA sequencing, as well as RNA and gene expression analysis and future technology for analysing proteins.

Learn about applications
ResourcesNewsExplore
Oxford Nanopore TechnologiesFully scalable, real-time DNA/RNA sequencing technology
Oxford Nanopore DiagnosticsLamPORE – rapid, low-cost, scalable detection of SARS-CoV-2
Contact
PCR Sequencing Kit
SQK-PSK004

Control over read length and compatible with targeted amplicon sequencing.

 Includes a Flow Cell Priming Kit
$599.00

Information

This kit is recommended for users who

  • have a low starting amount of DNA
  • want to optimise their sequencing experiment for throughput
  • require control over read length
  • are interested in targeted amplicon sequencing

Please note that to use this kit, you will need to purchase additional 3rd party reagents: see the "3rd Party Materials" tab for more detail.

The PCR Sequencing Kit is designed to prepare sequencing libraries when there is a limited amount of starting gDNA available.

The gDNA is fragmented in a Covaris g-TUBE. The sheared ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module, and then adapters which contain primer binding sites are ligated onto the prepared ends. An amplification step follows using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.

The kit involves ~50 mins pre-PCR work. The PCR step is variable (depends on the number of cycles, polymerase speed and template length). The standard protocol suggests starting with 100 ng of gDNA: less can be used, but it may be necessary to increase the number of PCR cycles. The post-PCR work is about 10 mins.

The PCR Sequencing Kit features:

FeatureProperty
Preparation time60 minutes + PCR
Input requirement<100 ng gDNA
PCR RequiredYes
FragmentationOptional
Read lengthEqual to fragment length post-PCR
Read type produced1D
Typical throughput*★★★
Associated protocolsPCR Sequencing Kit
Four-primer PCR (SQK-PSK004 or SQK-PBK004)
Multiplexing options• PCR Barcoding Kit
• Flow Cell Wash Kit
Pack size6 reactions
StabilityShipped at 2–8°C

Long-term storage -20°C

Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer.

The fragment length produced after PCR is dependent on the length of the material input into the PCR and is limited by the processivity of the DNA polymerase. This means that superior fragment lengths can be achieved with the PCR Sequencing Kit, compared to the Rapid PCR Barcoding Kit, which is limited to ~2 kb. Therefore this kit offers a solution to those who have limited amounts of starting material (i.e. need to do PCR) but require longer reads than are offered by the Rapid PCR Barcoding Kit.

2018 05 29 PSK004 v1 DS

Typical read-length histograms observed when preparing libraries with the PCR Sequencing Kit (A) and the Rapid PCR Barcoding Kit (B).

This kit also offers a method whereby users are able to tag their own specific amplicons with the same 5’ group, simplifying downstream post-PCR processing. This is done via a four-primer PCR. Users add a 5’ tail to their own primer sequences and this acts a priming site for a set of generic “outer” primers (supplied in the PCR Sequencing Kit) and these primers contain the 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.

Further considerations:

As well as being used to generate more material (where starting material is limiting), PCR can be used where sample purity is compromised (which can inhibit library preparation efficiency); PCR selects for molecules which have been successfully adapted, and generate more of them. The original impurity is lost or diluted. It is often observed that a sample which produces poor sequencing results can be improved by inclusion of PCR. In instances where less than 1 ng of input material is available, we recommend whole genome amplification.

A PCR Barcoding Kit is available for multiplexing up to 12 samples.

Shipping and logistics:

Flow cells and kits are shipped together at 2–8°C.

Upon receipt, please place the right product in the right long-term storage location.

Products are shipped to customers within the USA and EU Monday to Thursday. Shipments to Canada, Norway, Korea and Japan are expedited Monday to Wednesday; with Australia and New Zealand leaving our warehouses on a Friday. Shipments to the rest of the world are made on Mondays to allow the full working week for packages to arrive.

The delivery charges are calculated when a quote is raised or during checkout. Once an order is made, the delivery ID and delivery information can be tracked in the Store.

Equipment and consumables for PCR Sequencing Kit

Workflow

The PCR Sequencing Kit is designed to prepare sequencing libraries when there is a limited amount of starting gDNA available.

The gDNA is fragmented in a Covaris g-TUBE. The sheared ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module, and then adapters which contain primer binding sites are ligated onto the prepared ends. An amplification step follows using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.

The kit involves ~50 mins pre-PCR work. The PCR step is variable (depends on the number of cycles, polymerase speed and template length). The standard protocol suggests starting with 100 ng of gDNA: less can be used, but it may be necessary to increase the number of PCR cycles. The post-PCR work is about 10 mins.

2018 05 29 PSK004 workflow v1 DS

This kit also offers a method whereby users are able to tag their own specific amplicons with the same 5’ group, simplifying downstream post-PCR processing. This is done via a four-primer PCR. Users add a 5’ tail to their own primer sequences and this acts a priming site for a set of generic “outer” primers (supplied in the PCR Sequencing Kit) and these primers contain the 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters. Four-primer-PCR nonBC

Protocols

PCR Sequencing Kit
Four-primer PCR (SQK-PSK004 or SQK-PBK004)

To access the protocols, you will need to register for a Nanopore Community account.

What's in the box

The PCR Sequencing Kit contains sufficient reagents to generate 6 sequencing libraries. 2018 05 29 PSK004 contents v1 DS

NameAcronymCap colourNo. of vialsFill volume per vial (µl)
Whole Genome PrimersWGPGrey110
PCR AdapterPCABlue1140
Rapid AdapterRAPGreen110
Sequencing TetherSQTPurple110
Loading BeadsLBPink1360
Sequencing BufferSQBRed1300

The Flow Cell Priming Kit is also supplied. FLP

NameAcronymCap colourNo. of vialsFill volume per vial (μl)
Flush BufferFBBlue61,170
Flush TetherFLTPurple1200

3rd party materials

Consumables

  • Agencourt AMPure XP beads
  • NEBNext End repair / dA-tailing Module (E7546)
  • NEB Blunt/TA Ligase Master Mix (M0367)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • LongAmp Taq 2X Master Mix (e.g. NEB M0287)
  • 10 mM Tris-HCl pH 8.5 with 50 mM NaCl

Optional consumables

  • Covaris g-TUBE
  • Exonuclease I (NEB, M0293)

Equipment

  • Hula mixer (gentle rotator mixer)
  • Magnetic rack
  • Microfuge
  • Vortex mixer
  • Ice bucket with ice
  • Timer
  • Thermal cycler
  • P1000 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • P2 pipette and tips

Optional equipment

  • Agilent Bioanalyzer (or equivalent)
  • Qubit fluorometer (or equivalent for QC check)
  • Eppendorf 5424 centrifuge (or equivalent)

Compatibility

The PCR Sequencing Kit can be used together with:

Kits

  • Flow Cell Wash Kit (EXP-WSH004)

Flow cells

  • FLO-MINSP6
  • FLO-MIN106D

Devices

  • MinION Mk1B
  • MinION Mk1C
  • GridION