Post Digest Bead Cleans and Plate Reads

Dates Performed: June 22nd and 24th

June 22nd - Performed 1.5X bead cleans on Plates A,B,C,D,E June 24th - Plate Reader Quantification for Plates A,B,C,D,E June 30th - Qubit 16 samples that did not fit on plate reader

1. 1.5X Bead Clean After Digestion - this removes any left over enzyme and buffer in your wells and keeps all DNA Make PEG-NaCl if not made previously (Made PEG NACL on 6/21): PEG-NaCl: the salt solution that the beads are in, and this solution facilitates binding of the DNA to the beads. Keep freshly made PEG in the fridge when not in use, and keep it wrapped in foil. To make PEG, use a 50mL conical with: 25mL of 5M NaCl solution (use a 5mL pipette for this) 10g of Polyethylene gylcol 8000 (use a weigh boat, scooper, and scale by the microwave) Nuclease Free water up to 49mL Invert and vortex a few times, then mix on the orbital shaker for a while to combine If PEG-NaCl is from the fridge, take it out and let it get to room temp (similar to how you would with beads), this will take at least 60 min Take plate out of fridge and let get to room temp (takes about 5-10 min) Make a 50mL conical with fresh 80% ethanol: 40mL 100% ethanol and 10mL nuclease free water Make a trough/basin for PEG, waste, Ethanol, and nuclease-free water Calculate how much PEG you’ll need: 801.5 = 120ul. 120ul * number of samples Add that much PEG-NaCl plus a little extra into a basin. PEG-NaCl is very viscous, so pipette slowly when using it Use the multichannel to add 120ul of PEG-NaCl to each well of sample in your plate. Pipette slowly because it is viscous. Pipette mix at least 20 times because you want to mix the beads and everything very well. The volume in each well will basically be at its maximum, so be careful Once all of your sample wells have PEG, put the plate on the orbital shaker for 15 minutes at 200rpm to help bind the DNA to the beads After 15 minutes, transfer the plate to the white magnet rack with the metal posts, put this back on the shaker for at least 5 minutes. It will take a while for the beads to come to the magnet on this cleanup because the volume is so large. Be patient waiting for the solution to become clear and the beads to form a pellet on the side of the tube with the magnet When it’s ready, remove 190ul of the clear supernatant from each well using the multichannel and expel it into a waste trough. Slide the tips down the side of the wells opposite from the bead pellet Add 200ul 80% ethanol to each well Remove 200ul of the supernatant from each well, sliding the tips down the side of the wells opposite from the bead pellet Add 180ul 80% ethanol to each well Remove 200ul of the supernatant from each well, sliding the tips down the side of the wells opposite from the bead pellet Make sure all liquid is out of each well by going back in with the multichannel for the p20 pipette set to 20ul and suck up any extra liquid at the bottom of each well Let wells sit open to “dry” for 2 minutes. You want extra ethanol to evaporate but you don’t want the beads to dry until cracked Use a basin with 10mM Tris HCl (33number of samples + a little extra) Resuspend the beads in 33ul of 10mM Tris HCl. Pipette up and down to get the bead pellet off as best you can Keep the beads in!!! Cover the plate with a foil seal and store in the 4 degree fridge if not quantifying that day If you are quantifying, cover with a silicon pad and keep on an ice bucket until ready

2. Quantify Digested DNA Use the Biotium AccuBlue Plate Reader Protocol You don’t need to use all the standards, the concentration will likely not be over 50ng/ul If you do the whole row A of standards, this is a good suggestion: 2 0ng/ul 2 2ng/ul 2 6.25ng/ul 2 12ng/ul 2 25ng/ul 1 50ng/ul 1 100ng/ul It’s best to use the same plate layout as you have, if you have blank wells and you don’t need to use the whole 96 well plate for the assay, just leave those wells blank on the assay Follow instructions in the protocol for generating a standard curve and calculating the concentration of DNA in each well

Combined plates B/C and D/E for reading. Row D from plates C and E were not read on plate reader. Qubit results below for those samples:

DROPPING SAMPLES 838, 428, 389 from library prep due to low DNA concentration