Section 1: Enzymatic DNA Fragmentation (Manually)
Ester Kalef-Ezra, Christos Proukakis, Ben Harvey, Katherine Roper
Abstract
This protocol details manual enzymatic DNA Fragmentation prior to Section 2: NGS library preparation for sequencing.
Attachments
Steps
Enzymatic DNA Fragmentation (Manually)
Group the samples scWGA-products of interest for library preparation based on their size length to require the same fragmentation time. The recommended fragmentation times per amplicon are suggested on Table 1.
Table 1 . Recommended fragmentation time based on amplicon size.
| A | B |
|---|---|
| scWGA products peak size (bp) | Fragmentation Time (min) |
| 400-450 | 3 |
| 450-500 | 4 |
| 500-550 | 5 |
| 550-650 | 6 |
| 650-750 | 7 |
| 750-900 | 8 |
| 1000-1100 | 10 |
| >10.000 | 15 |
Calculate the amount of input material needed from each scWGA-product to have input of 10-200ng DNA in a total volume of 7µL.
Thaw the vial of 5X SureSelect Fragmentation Buffer On ice, vortex, then keep On ice.
Thaw a PCR plate cooler @Room temperature for 0h 5m 0s.
Pre-label PCR tubes/strips to be used for the enzymatic DNA fragmentation reaction.
Place the prelabelled PCR tubes/strips/plate in the PCR plate cooler.
Dilute 10-200ng of each scWGA-product with dH2O up to a final volume of 7µL in the PCR tubes/strips.
Pre-program a thermal cycler as on Table 2. If required, use a reaction volume setting of 10µL and lid temperature @60-65°C.
Table 2 . Thermal cycler program for Enzymatic Fragmentation (with the heated lid ON).
| A | B | C |
|---|---|---|
| Step | Temperature (ºC) | Time (min) |
| 1 | 37 | The max need based on Table 1 |
| 2 | 65 | 5 |
| 3 | 4 | hold |
Prepare the appropriate volume of Fragmentation master mix by combining the reagents as on Table 3 in 0.2ml or 1.5 ml tube.
Table 3 . Fragmentation master mix.
| A | B |
|---|---|
| Reagent | 1 reaction |
| 5X SureSelect Fragmentation Buffer | 2 μl |
| SureSelect Fragmentation Enzyme | 1 μl |
| Total | 3 μl |
Mix well by pipetting up and down 20 times or seal the tube and vortex at high speed for 5–10 sec.
Spin briefly to remove any bubbles and keep On ice.
Add 3µL of the Fragmentation master mix to each sample well containing 7µL of input DNA.
Mix well by pipetting up and down 20 times or cap the wells and vortex at high speed for 5–10 seconds.
Spin the samples briefly and place on the PCR plate cooler.
Place the tubes/strip in the thermal cycler and start the ‘Enzymatic Fragmentation’ program.
When the program reaches the 4°C Hold step, remove the samples from the thermal cycler.
Add 40µL of nuclease-free water to each sample and place the samples On ice.
