Pooling Plate C - Pools 4 and 5

Pooling Plate C then doing 2 1.5X cleans

Date Performed: December 16th, 2021

Plate C Pooling Notes

  • Plate C = pools 4 and 5
  • When pool 4 divided into 4 tubes, each had about 350ul of sample - used 525ul beads for first 1.5X clean

  • When pool 5 divided into 4 tubes, each had about 350ul of sample - used 525ul beads for first 1.5X clean

    Pool 4 Qubit Results after First 1.5X Clean - Qubit Assay using AcuGreen Reagent

    Sample Run 1 Run 2
    S1 420.97  
    S2 112253.24  
    Tube 1 5.58 5.56
    Tube 2 6.88 6.86
    Tube 3 6.62 6.74
    Tube 4 6.58 6.54

    Qubit Results after Second 1.5X Clean - Qubit Assay using AcuGreen Reagent

    Sample Run 1 Run 2
    S1 416.5  
    S2 110423.40  
    Tube 1 14.4 14.2

    Pool 5 Qubit Results after First 1.5X Clean - Qubit Assay using AcuGreen Reagent

    Sample Run 1 Run 2
    S1 421.15  
    S2 110696.73  
    Tube 1 5.66 5.70
    Tube 2 7.40 7.46
    Tube 3 7.70 7.58
    Tube 4 7.34 7.34

    Qubit Results after Second 1.5X Clean - Qubit Assay using AcuGreen Reagent

    Sample Run 1 Run 2
    S1 415.57  
    S2 113120.13  
    Tube 1 14.7 14.5

Followed Pooling Samples and 2 1.5X Clean-Ups (Step 12) from ddRAD Protocol

  • Take BEADS out of the fridge for ~30 minutes to get to room temperature and make new 80% Ethanol
  • For each pool: Transfer the full 40μl of ligation reaction (times the number of wells you are pooling), including the beads to a single 2mL tube. It might help to pipette mix first to make sure to transfer them all. At this stage, do not combine samples from different pools together. It is best to do the pooling and clean ups 1 pool at a time to avoid confusion
  • For each pool: split the 2mL tube into 4 1.5mL tubes with equal volume in each, using a p200 pipette 50μl repeatedly to make sure each tube has the same volume. You can calculate an estimate of the volume in each of the 4 tubes, but usually there has been some evaporation in some wells, so each is not exactly 40ul when you combine them together (you’ll notice this in the step above)
  • Add 1.5 times the volume of the 4 tubes of BEADS to each aliquot tube and pipette to mix. This number will change depending on the number of samples per pool
  • Incubate the 4 tubes on shaker for 15 minutes
  • Place tubes on the magnet bar (long one) and wait until the solution goes clear. You can pipette to encourage the beads to move to the magnet
  • Remove as much clear liquid as you can without removing any beads (10-20ul less than the volume in there)
  • Add 1000μl 80% EtOH to each tube while keeping it on the magnet
  • Remove 1000μl of the EtOH from the tubes and discard
  • 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, to let the ethanol evaporate but not letting the beads completely dry out
  • Remove tubes from magnet and add 40μl of 10mM Tris HCl to each tube, mix by pipetting, and incubate for 5 minutes on the shaker to make sure all the beads are separated from the tube wall
  • Stop! Qubit these tubes to make sure you did not lose DNA. Use Qubit Protocol
  • You should have about 100ng * how many samples per pool total DNA. To calculate total DNA, multiply the qubit value x 4 x 40.
  • Once confirming no dramatic DNA loss, recombine the 4 separated tubes back together into one tube (1 for each pool), this can be one of the tubes you already have samples in
  • Add 1.5X BEADS (240μl) to each tube, mix by pipetting, and incubate on a shaker for 5 minutes
  • Put the tubes on the magnet, 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 from the tubes and discard
  • 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, to let the ethanol evaporate but not letting the beads completely dry out
  • Remove tubes from magnet and add 60μl of 1X TE buffer to each tube and pipette to mix. Incubate for 5 minutes on the shaker
  • Put tubes back onto the magnet and wait until the liquid goes clear, then remove supernatant and transfer to a new strip tube and keep on ice
  • Repeat these steps for all pools that have gone through the ligation test and are ready to be combined. It is best to cleanup each pool 1 at a time to avoid confusion
  • Qubit the pools after the two clean ups to make sure that there is DNA going into the size selection.
  • These tubes can now be frozen.
Written on December 16, 2021