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:
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.
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.
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.
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.
What's in the box
The PCR Sequencing Kit contains sufficient reagents to generate 6 sequencing libraries.
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (µl) |
|---|
| Whole Genome Primers | WGP | Grey | 1 | 10 |
| PCR Adapter | PCA | Blue | 1 | 140 |
| Rapid Adapter | RAP | Green | 1 | 10 |
| Sequencing Tether | SQT | Purple | 1 | 10 |
| Loading Beads | LB | Pink | 1 | 360 |
| Sequencing Buffer | SQB | Red | 1 | 300 |
The Flow Cell Priming Kit is also supplied.
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (μl) |
|---|
| Flush Buffer | FB | Blue | 6 | 1,170 |
| Flush Tether | FLT | Purple | 1 | 200 |
Compatibility
The PCR Sequencing Kit can be used together with:
Kits
- Flow Cell Wash Kit (EXP-WSH004)
Flow cells
Devices
- MinION Mk1B
- MinION Mk1C
- GridION
