产品说明书: KAPA Library Amplification KitVendor   

Product Description

KAPA Library Amplification Kits for Illumina platforms are designed for the amplification of next-generation sequencing libraries prepared for Illumina sequencing.

In order to maximize sequence coverage uniformity, it is critical to minimize library amplification bias. KAPA HiFi DNA Polymerase is designed for low-bias, high-fidelity PCR, and is the reagent of choice for NGS library amplification^1,2,3,4. KAPA Library Amplification Kits include KAPA HiFi HotStart ReadyMix (2X), a ready-touse PCR mix comprising all the components for library amplification— except primers and template. Kits also include Library Amplification Primer Mix (10X), designed for the high-efficiency amplification of Illumina libraries flanked by adapters containing the P5 and P7 flow cell sequences.

1. Oyola, S.O., et al., BMC Genomics 13, 1 (2012).
2. Quail, M.A., et al., Nature Methods 9, 10 (2012).
3. Quail, M.A., et al., BMC Genomics 13, 341 (2012).
4. Ross, M.G., et al., Genome Biology 14, R51 (2013).
   

Product Applications

KAPA Library Amplification Kits for Illumina platforms are suited for high-efficiency, high-fidelity, low-bias amplification of libraries prior to Illumina sequencing. This includes libraries prepared for:
• whole-genome shotgun sequencing
• targeted sequencing (pre- and post-capture amplification)
• amplicon sequencing
• ChIP-seq
• RNA-seq.

Library Amplification Protocol

1. Library Amplification
   
   Library Amplification Primer Mix (10X) (KK2623), sold separately, is designed to eliminate or delay primer depletion during library amplification reactions performed with KAPA HiFi HotStart ReadyMix (2X). The primer mix is suitable for the amplification of all Illumina libraries flanked by the P5 and P7 flow cell sequences. Primers are supplied at a 10X concentration of 20 μM each, and have been formulated as previously described. User supplied primer mixes may be used in combination with incomplete or custom adapters. For guidelines on the formulation of user-supplied library amplification primers, please contact Technical Support at sequencing.roche.com/support.
   1.1

   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 0.5-2 μM.
   
   
   1.2

   Mix thoroughly and centrifuge briefly.
   1.3

   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.

   *The optimal cycling number will depend upon the volume and concentration of adapter-ligated, size separated, purified library DNA added to each enrichment PCR reaction.

   
   
   1.4

   Proceed directly to Post-amplification Cleanup (step 2).
   
   
2. Post-amplification Cleanup
   2.1

   In the library amplification plate/tube(s), perform a 1X bead-based cleanup by combining the following:
   
   
   
   2.2

   Mix thoroughly by vortexing and/or pipetting up and down multiple times.
   2.3

   Incubate the plate/tube(s) at room temperature for 5-15 min to bind DNA to the beads.
   2.4

   Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
   2.5

   Carefully remove and discard the supernatant.
   2.6

   Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
   2.7

   Incubate the plate/tube(s) on the magnet at room temperature for ≥ 30 sec.
   2.8

   Carefully remove and discard the ethanol.
   2.9

   Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
   2.10

   Incubate the plate/tube(s) on the magnet at room temperature for ≥ 30 sec.
   2.11

   Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
   2.12

   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.
   2.13

   Remove the plate/tube(s) from the magnet.
   2.14

   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.
   2.15

   Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads.
   2.16

   Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
   2.17

   Transfer the clear supernatant to a new plate/tube(s). Store purified, amplified libraries at 2 °C to 8 °C for 1 – 2 weeks, or at -15 °C to -25 °C.