1X Bead clean to concentrate DNA into smaller volume before sonication

Completed April 14, 2021

Before sonicating the DNA, a few samples need to have the DNA concentrated into a smaller volume. This is necessary because some samples had too little DNA to achieve 500 ng of DNA in 51 ul (volume needed for sonication).

• Made fresh 80% EtOH
• Took KAPA Pure Beads out of fridge beforehand to warm to room temp for about 30 minutes
• Vortex and spin down DNA samples
• For sample MCD_10, combine MCD_10 M E2 and MCD_10 G E2 into new 1.5 mL tube
• In new 1.5 mL tubes, add appropriate volume of sample (see column 3 in table above)
• Add appropriate volume of KAPA Pure Beads (see column 4 in table above) to each sample and pipette up and down 10 times to mix (avoid bubbles)
• Place tubes on shaker at room temp for 15 minutes - shaker set to 200 rpm’s
• After 15 minute incubation, place tubes on magnet plate and remove supernatant from tubes when it was fully clear not disturbing the beads
  • Dispose of liquid in liquid waste beaker
• Add 200 μl of 80% EtOH to each tube while still the magnet not disturbing the beads
• Remove supernatant from each tube on the magnet plate without disturbing the beads
• Add 200 μl of 80% EtOH to each tube while still the magnet not disturbing the beads
• Remove ALL the supernatant from each tube on the magnet plate without disturbing the beads. Extra EtOH blobs were removed with p20 pipette tips
• Resuspend beads in 30 μl of 10 mM Tris HCl pH 8 and incubate tubes at room temp for 5 minutes
  • Did not put on shaker this time
• Place tubes on magnet plate and transfer supernatant when clear to new labeled 1.5 mL tubes
• 1 ul of each sample used for Qubit

29 ul remaining in each sample

Qubit dsDNA BR assay

• Followed Qubit protocol for BR DNA

A lot of DNA was lost during the bead clean, so all of these samples have less than 500 ng total. Thankfully I have replicate extracted DNA for all these samples, so I can use those to get 500 ng of DNA and move onto sonication.