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- Mix thoroughly by pipetting up and down five times, then
transfer master mixes to two wells of a 96-well PCR plate
(Table 1 ).
- The tubes with master mix and DNA were placed in a thermal
cycler (Fig. 1 ). They will be activated according to the thermal
program outlined in Table 2 to amplify target genomic regions
(see Note 2).
- At the completion of amplification, new strip tubes were
obtained for all the samples.
- Amplification products for each primer pool were combined
into one tube and the total volume in each tube was 20 μL.
- To each combined amplicon pool, 2 μL of FuPa reagent was
added to partially digest primers for ligation. Total reaction
volume was 22 μL.
- The mixture was pipetted up and down five times to mix and
the strip tubes were placed in a thermal cycler and temperature
was performed according to the program as indicated in
Table 3. We can make this step as STOPPING POINT and
store plate at − 20 °C for longer periods.
Table 1
Master mix for library preparation
Component Volume (μL)
5× Ion Ampliseq HiFi Master Mix 2
2× Ion Ampliseq Primer Pool 5
G DNA, 10 ng 1
Nuclease-free water 2
Total 10
Table 2
Thermal program for amplification
Stage Step Temperature (°C) Time Cycles
Hold Activate the
enzyme
99 2 min 1
Cycles Denature 99 15 s
Anneal and
extend
60 4 min 18
Hold 10 Hold 1
Next Generation Sequencing and Esophageal Adenocarcinoma
