TITLE: Section 1: Enzymatic DNA Fragmentation (Manually)
AUTHORS: Ester Kalef-Ezra, Ben Harvey, Katherine Roper, Christos Proukakis

[DESCRIPTION]
This protocol details manual enzymatic DNA Fragmentation prior to Section 2: NGS library preparation for sequencing.


[STEPS]
SECTION: Enzymatic DNA Fragmentation (Manually)
1. 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.
 
      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  

SECTION: Enzymatic DNA Fragmentation (Manually)
2. Calculate the amount of input material needed from each scWGA-product to have input of 10-200 ng DNA in a total volume of 7 µL. 
 

SECTION: Enzymatic DNA Fragmentation (Manually)
3. Thaw the vial of 5X SureSelect Fragmentation Buffer on ice, vortex, then keep on ice.

SECTION: Enzymatic DNA Fragmentation (Manually)
4. Thaw a PCR plate cooler @Room temperature for 5 min.
SECTION: Enzymatic DNA Fragmentation (Manually)
5. Pre-label PCR tubes/strips to be used for the enzymatic DNA fragmentation reaction.
SECTION: Enzymatic DNA Fragmentation (Manually)
6. Place the prelabelled PCR tubes/strips/plate in the PCR plate cooler.
SECTION: Enzymatic DNA Fragmentation (Manually)
7. Dilute 10-200 ng of each scWGA-product with dH2O up to a final volume of 7 µL in the PCR tubes/strips.
 

SECTION: Enzymatic DNA Fragmentation (Manually)
8. Pre-program a thermal cycler as on Table 2. If required, use a reaction volume setting of 10 µL and lid temperature @60-65 °C.

Table 2. Thermal cycler program for Enzymatic Fragmentation (with the heated lid ON).  
      Step  Temperature (oC)  Time (min)    1  37  The max need based on Table 1    2  65  5    3  4  hold  

SECTION: Enzymatic DNA Fragmentation (Manually)
9. 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. 
      Reagent  1 reaction    5X SureSelect Fragmentation Buffer  2 μl    SureSelect Fragmentation Enzyme  1 μl    Total  3 μl  

SECTION: Enzymatic DNA Fragmentation (Manually)
10. Mix well by pipetting up and down 20 times or seal the tube and vortex at high speed for 5–10 sec.
SECTION: Enzymatic DNA Fragmentation (Manually)
11. Spin briefly to remove any bubbles and keep on ice.

SECTION: Enzymatic DNA Fragmentation (Manually)
12. Add 3 µL of the Fragmentation master mix to each sample well containing 7 µL of input DNA.

SECTION: Enzymatic DNA Fragmentation (Manually)
13. Mix well by pipetting up and down 20 times or cap the wells and vortex at high speed for 5–10 seconds.
SECTION: Enzymatic DNA Fragmentation (Manually)
14. Spin the samples briefly and place on the PCR plate cooler.
SECTION: Enzymatic DNA Fragmentation (Manually)
15. Place the tubes/strip in the thermal cycler and start the ‘Enzymatic Fragmentation’ program.
 

SECTION: Enzymatic DNA Fragmentation (Manually)
16. When the program reaches the 4 °C Hold step, remove the samples from the thermal cycler.
SECTION: Enzymatic DNA Fragmentation (Manually)
17. Add 40 µL of nuclease-free water to each sample and place the samples on ice.