Pools 1 and 2 PCR, 1.5X Clean, and 2X Qubit

Date Performed: January 19th, 2022

PCR and Index Addition USE FILTER TIPS FOR ALL PIPETTING

• For each pool you are going to set up 6 PCR reactions. This minimizes possible PCR biases but also amplifies the libraries to a good amount. If you are amplifying more than one pool at a time (say 5), it can be easier to carry out all the reactions on a 96 well plate instead of individual strip tubes
• Set up a plate or however many tubes you’ll need to have 6 wells/tubes per pool. Label A, B, C etc as these are very temporary
• Thaw size-selected samples, ready mix, and 50X, 70X primers on ice. Vortex and spin down all before using
• Each pool needs a master mix because each pool gets their own 50X-70X primer pair
• Make a master mix for each pool (ex for 5 pools, you would make 5 mixes, each with their specific primer pair):
  • 10µl of 2X KAPA HiFi Hot Start Ready Mix * 6.1 = 66ul
  • 4µl water * 6.1 = 26.4ul
  • .5μl 20uM PCR primer 50X *6.1 = 3.3ul
  • .5μl 20uM PCR primer 70X *6.1 = 3.3ul
• Vortex and spin down the master mixes
• Pipette 15ul of each master mix into their planned 6 tubes or wells
• Add 5ul of DNA from a size-selected pool to their respective wells, and repeat for the other pools
• Cover the plate with a foil seal, vortex to mix, and spin down
• Put in the Thermocycler for amplification, 12 cycle RAD PCR program under JONP login
• Samples can be frozen afterwards, or continue to clean-up

Notes on PCR

Pool 1 Index Pair = 502/702 Pool 2 Indx Pair = 505/705

1.5X Cleanup after PCR

• Take out KAPA Pure Beads ~30 minutes before use and make fresh 80% ethanol
• Combine the 6 reactions into a single 1.5 mL tube, for a total of 120μl
• Add 1.5X of KAPA Pure Beads (180μl) to each pooled tube, mix by pipetting, and incubate on a shaker for 15 minutes
• Put the tubes on the long magnet rack, wait until the liquid becomes clear, and remove as much of the clear liquid as you can
• Add 1000μl 80% EtOH to each tube while keeping it on the magnet
• Remove 1000μl of the EtOH
• Add 1000μl 80% EtOH to each tube
• Remove ALL EtOH carefully from each tube, use the p20 or p200 to remove any excess ethanol
• Let samples sit for ~5 minutes, letting the ethanol evaporate, but the beads not dry out
• Take tubes of the magnet stand and elute the DNA in 35μl 10mM Tris HCl and incubate shaking for 5 minutes
• Place the tubes back in the magnet, and wait until the liquid goes clear. Remove all liquid and put into new strip tubes: this is your library!

Qubit Results