Amplicon-based Library Construction for Next Generation Sequencing Using the EzMate Automated Pipetting System
Benefits
- Obtain the benefit of simple and high-qualified NGS sample library, also prevents manual pipetting errors with EzMate Automated Pipetting System
- Keep the polymerase at an appropriate cool temperature by using EzMate 601s Automated Pipetting System
Introduction
Seeing Bioscience is a company that develops the technology for diagnosis of disease using molecular biology and provides comprehensive molecular biology services including next generation sequencing (NGS). In the NGS preparation, amplicon-based library construction is popular to analyze and trace targeted gene sequence. It is common to prepare hundreds of amplicon libraries for an amplicon-based NGS service case. In this application note, we demonstrate that the EzMate Automated Pipetting System is an ideal tool for the amplicon-based NGS preparation. It does well in amplicon library construction and provides a cool environment for Taq polymerase.
Materials
Reagents and Consumables:
- FFPE DNA samples
- Nested PCR primers (outer)
- Barcode PCR primers (inner)
- Recipe of PCR master mix: Betaine, dNTP, 10X Supertherm GOLD buffer, Supertherm GOLD Taq polymerase
- 96-well and 384-well PCR plates
Equipment:
- EzMate 401 & 601s Automated Pipetting System
- 8-channel, 50µl Automatic Pipetting Module (APM50-8)
- CoolBlock 384 adapter for 384-well PCR plate
- General laboratory equipments
EzMate 401
EzMate 601s
Methods
- Use a manual pipette to mix PCR master mix and outer PCR primers in a 2ml screw cap tube. Transfer the mixture to one column of a 96‐well PCR microplate.
- Repeat step 1 to transfer 4 different kinds of PCR mixture, each with one pair of outer PCR primers to a 96‐well PCR microplate.
- Place the 96‐well PCR microplate with mixture on area C of the EzMat 601s. Transfer the 9μl PCR mixture with outer primers to a 384‐well PCR microplate on area A (Fig. 1).
Fig. 1 Labware Layout of the EzMate 601s.
4. Place the 384‐well PCR microplate with PCR mixture on area A and the 96‐well PCR microplates with 96 DNA samples on area C of the EzMat 601s. Transfer the 1μl DNA samples to a 384‐well PCR microplate on area A (Fig. 2).
Fig. 2 Labware Layout of the EzMate 401.
5. After the PCR assay is completed, place the 384‐well PCR microplate with 384 PCR products (which will be used as DNA samples to perform the PCR assay with inner PCR primers) on area C of the EzMate 401.
6. Repeat steps 1~3, but add inner primers to prepare the PCR master mix with inner PCR primers for the second PCR.
7. Transfer the 1μl PCR products in the 384‐well PCR microplates on area C to the 384‐well PCR microplate on area A of the EzMate 401. Then, perform the PCR assay.
8. Repeat above steps for all the other samples and primer pairs.
Results
The data in Fig. 3 indicates that the EzMate series can handle fatiguing pipetting tasks in amplicon library construction and performs with high accuracy and good precision.
By implemented the EzMate series to do the amplicon-based library construction, user can obtain a high-quality sequencing library which is very important for NGS, and prevent manual pipetting errors so as to save time and money.
In this experiment, the EzMate 601s with Active Cooling and Heating Module (ACHM) and the CoolBlock adapter keeps the polymerase at an appropriate cool temperature that is important for PCR amplification and sequencing.
Fig. 3 Analysis of PCR products using agarose gel.
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