PCR Sequencing Kit
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
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.
Key features:
Prep time | PCR + 60 minutes |
Input amount | 100 ng dsDNA |
Read length | = fragment length post-PCR |
Typical throughput | 2-3+ Gb in 6 hours, 8+ Gb in 48 hours per flow cell on MinION/GridION |
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.
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