产品说明书: KAPA Hyper Prep C (96rxn)Vendor   

Library Construction Protocol

Note: This protocol does not include size selection. Please refer to Appendix 1 for detailed double-sided size selection protocols.

1. End Repair and A-tailin
   1.1

   Assemble each end repair and A-tailing reaction in a tube or well of a PCR plate as follows:
   
   *The buffer and enzyme mix should preferably be pre-mixed and added in a single pipetting step. Premixes are stable for ≤ 24 hrs at room temperature, for ≤ 3 days at 2 °C to 8 °C, and for ≤ 4 weeks at -15 °C to -25 °C.
   
   
   1.2

   Vortex gently and spin down briefly. Return the plate/tube(s) to ice. Proceed immediately to the next step.
   1.3

   Incubate in a thermocycler programmed as outlined below:
   
   *A heated lid is required for this incubation. If possible, set the temperature of the lid at 85 °C, instead of the usual ~105 °C.
   **If proceeding to the adapter ligation reaction setup without any delay, the reaction may be cooled to 20 °C instead of 4 °C.
   
   
   1.4

   Proceed immediately to Adapter Ligation (step 2). 
   
   
2. Adapter Ligation
   2.1

   Dilute adapter stocks to the appropriate concentration, as outlined in Table 2 (p. 5).
   2.2

   In the same plate/tube(s) in which end repair and A-tailing was performed, assemble each adapter ligation reaction as follows:
   
   *The water, buffer and ligase enzyme should preferably be premixed and added in a single pipetting step. Premixes are stable for ≤ 24 h at room temperature, for ≤ 3 days at 4 °C, and for ≤ 4 weeks at -20 °C.
   
   
   2.3

   Mix thoroughly and centrifuge briefly.
   2.4

   Incubate at 20 °C for 15 min. 
   Note: to achieve higher conversion rates and library yields, particularly for low-input samples, consider increasing the ligation time – to a maximum of 4 h at 20 °C, or overnight at 2 °C to 8 °C. Please note that longer ligation times may lead to increased levels of adapter-dimer. Adapter concentrations may have to be optimized if ligation times are extended significantly.
   
   2.5

   Proceed immediately to the next step.
   
   
3. Post-ligation Cleanup
   3.1

   In the same plate/tube(s), perform a 0.8X bead- based cleanup by combining the following:
   
   
   
   3.2

   Mix thoroughly by vortexing and/or pipetting up and down multiple times.
   3.3

   Incubate the plate/tube(s) at room temperature for 5-15 min to bind DNA to the beads.
   3.4

   Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
   3.5

   Carefully remove and discard the supernatant.
   3.6

   Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
   3.7

   Incubate the plate/tube(s) on the magnet at room temperature for ≥ 30 sec.
   3.8

   Carefully remove and discard the ethanol.
   3.9

   Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
   3.10

   Incubate the plate/tube(s) on the magnet at room temperature for ≥ 30 sec.
   3.11

   Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
   3.12

   Dry the beads at room temperature for 3-5 min, or until all of the ethanol has evaporated. 
   Caution: over-drying the beads may result in reduced yield.
   3.13

   Remove the plate/tube(s) from the magnet.
   3.14

   Thoroughly resuspend the beads:
   •in 25 µL of elution buffer (10 mM Tris-HCl, pH 8.0-8.5) to proceed with Library Amplification (step 4), or
   •in 55 µL of elution buffer (10 mM Tris-HCl, pH 8.0-8.5) to proceed with double-sided size selection (Appendix 1).
   
   3.15

   Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads.
   3.16

   Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
   3.17

   Transfer the clear supernatant to a new plate/tube(s):
   •to proceed with Library Amplification (step 4), transfer 20 µL of supernatant, or
   •to proceed with double-sided size selection (Appendix 1), transfer 50 µL of supernatant.
   
   
4. Library Amplification
   Note: Please refer to Important Parameters: Library Amplification (pp. 6-7) and the KAPA NGS Library Preparation Technical Guide for more information on optimizing library amplification.
   
   
   4.1

   Assemble each library amplification reaction as follows:
   
   *Or another, suitable 10X library amplification primer mix. The recommended final concentration of each primer in the library amplification reaction is 0.5-4 µM. Also refer to Important Parameters: Library Amplification (p. 6).
   
   
   4.2

   Mix thoroughly and centrifuge briefly.
   4.3

   Amplify using the following cycling protocol:
   
   *Optimization of the annealing temperature may be required for non- standard (i.e., other than Illumina TruSeq) adapter/primer combinations.
   
   
   4.4

   Proceed directly to Post-amplification Cleanup (step 5).
   
   
5. Post-amplification Cleanup
   5.1

   In the library amplification plate/tube(s) perform a 1X bead-based cleanup by combining the following:
   
   
   
   5.2

   Mix thoroughly by vortexing and/or pipetting up and down multiple times.
   5.3

   Incubate the plate/tube(s) at room temperature for 5-15 min to bind DNA to the beads.
   5.4

   Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
   5.5

   Carefully remove and discard the supernatant.
   5.6

   Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
   5.7

   Incubate the plate/tube(s) on the magnet at room temperature for ≥ 30 sec.
   5.8

   Carefully remove and discard the ethanol.
   5.9

   Keeping the plate/tube(s) on the magnet, add 200 µL of 80% ethanol.
   5.10

   Incubate the plate/tube(s) on the magnet at room temperature for ≥ 30 sec.
   5.11

   Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
   5.12

   Dry the beads at room temperature for 3-5 min, or until all of the ethanol has evaporated.
   Caution: over-drying the beads may result in reduced yield.
   5.13

   Remove the plate/tube(s) from the magnet.
   5.14

   Thoroughly resuspend the beads in an appropriate volume of elution buffer (10 mM Tris-HCl, pH 8.0-8.5) or PCR-grade water. Always use PCR-grade water if proceeding to target capture. Note: If proceeding with a second post-amplification cleanup, or double-sided size selection (Appendix 1), resuspend the beads in 55 µL of elution buffer.
   5.15

   Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads.
   5.16

   Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
   5.17

   Transfer the clear supernatant to a new plate/tube(s) and proceed with size selection (refer to Appendix 1), library QC, target capture or sequencing, as appropriate. Store purified, amplified libraries at 2 °C to 8 °C for 1-2 weeks, or at -15 °C to -25 °C.