PCR and Index Addition Pool 1, 1.5X clean pools 1,3,4,5, qubit, gel

PCR for Pool 1, 1.5X clean, qubit, gel pools 1,3,4,5

Date Performed: February 1st, 2022

PCR and Index Addition Pool 1 Notes on PCR
Pool 1 Index Pair = 502/702

1.5X Bead Clean on Pools 1,3,4,5

  • 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!

2X Qubit for each pool

Sample Run 1 Run 2
S1 440.54  
S2 107362.69  
Pool 1 4.94 4.90
Pool 3 7.32 7.26
Pool 4 7.72 7.78
Pool 5 10.9 10.8
Sample Run 1 Run 2
S1 413.73  
S2 108102.91  
Pool 1 5.24 5.24
Pool 3 7.40 7.38
Pool 4 7.98 7.80
Pool 5 10.6 10.7

12 cycle PCR on 1ul from size selected pools 1,3,4,5 for gel

Each PCR Tube for Pools 1,3,4,5 * 1 ul sample
* 10ul Kapa Hifi * .5 ul 505 primer * .5 ul 705 primer * 8ul nuclease free water

Pool 1 = 502/702 Pool 3 = 506/706 Pool 4 = 507/707 Pool 5 = 508/708

gel

Written on February 1, 2022