Two 1.5X bead cleans and plate pooling

Dates Performed: July 8th (Plates) July 13th (1st Bead Clean Plate B) July 15th (2nd Bead Clean Plate B, C, D, E)

Step 12: Pooling Samples and 2 1.5X Clean-ups
Take the PEG-NaCl 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 multichannel pipette mix first (set to 25ul) 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 100μl (then 50ul and so on) repeatedly to make sure each tube has the same volume. Keep a tally of how many ul are separated out. 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 PEG-NaCl 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
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 PEG-NaCl (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. These tubes can now be frozen\

July 8th
Plate A was divided into 4 tubes of ~350ul each\

July 13th
Plate B was divided into 4 tubes of ~350ul each. Only completed first bead clean up to recombining back in tube A. Did not put samples on orbital shaker for five minutes after adding the 40ul of Tris HCL but appeared that all beads were removed from walls.

July 15th
Pooled Plates B, C, D, and E Plate B only needed one more bead clean Plate C was divided into 4 tubes of ~330ul each
Plate D was divided into 4 tubes of ~350ul each
Plate E was divided into 4 tubes of ~350ul each