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Indexing PCR

This protocol has been adapted from the protocols of Jordan Bisanz Laboratory at the Pennsylvania State University.

Prepare Your Workspace

  • Turn on the PCR Clean Hood blower and white light.
  • Clean the cabinet with 70% ethanol including work surface and walls.
  • Turn the dial and run the UV light for at least 10 minutes.

⚠️ You cannot expect the glass to protect you from UV exposure. You can be in a different part of the lab while the light is running, but do not loiter in front of the cabinet.

  • Once the timer is up and the UV light turns off, you are ready to begin.

Remember, before placing any objects in the hood, they must be sprayed with 70% ethanol.

Gather Materials

  • 96-well PCR Plates
  • DMSO
  • KAPA HiFi PCR kit (KAPA KK2502)
  • Indexing Primers
  • Nuclease free water
  • Primary PCR amplicons
  • Plate Films

Protocol

  1. Thaw KAPA HiFi PCR Kit on ice.
  2. Dilute primary PCR amplicons 1:100 in nuclease free water.
  3. Make indexing PCR master mix as outlined in the table below:

Table 1. Indexing PCR Master Mix

Reagent 1 Reaction (20uL) 110 Reactions (Full Plate)
5X KAPA HiFi Buffer 4uL 440uL
10mM dNTPs 0.6uL 66uL
DMSO 1uL 110uL
KAPA HiFi Polymerase 0.4uL 44uL
Total 6.0uL 660uL
  1. To a new 96-well PCR Plate, for each well add:
  • 6uL of Master Mix
  • 4uL of Indexing Primer
  • 10uL of Diluted PCR Amplicons
  1. Vortex and centrifuge plate. Place in thermocycler using the indexing amplification parameters as outlined in Table 2.

Table 2. Indexing Amplification Parameters.

Cycle Temperature Time
Initial (Denaturation) 95˚C 5 minutes
10 Cycles:
Denature 98˚C 20 seconds
Anneal 55˚C 15 seconds
Extend 72˚C 60 seconds
Hold at 4˚C