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

Completed April 19, 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).

Sample	Description	Volume for 500 ng (ul)	Volume of Beads (ul)
GHP_2	Third extraction E2	60	60
MCD_10	Third extraction E2 gill tissue	150	150

• 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, add 3.2G sample volume (all) to M & G E2 combined sample (made previously), for a total volume of 294 uL
• 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 20 μl of 10 mM Tris HCl pH 8 for sample GHP_2 and 51 uL for sample MCD_10
• Incubate tubes at room temp on shaker for 5 minute
• Place tubes on magnet plate and transfer supernatant when clear to new labeled 1.5 mL tubes

Some samples needed a little more DNA to reach 500 ng, so I took DNA from other extractions/elutions (within the same sample) to reach concentration

Sample	Sample Type 1 Description	Sample Type 2 Description	Type 2 Volume to add for 500 ng (ul)	Total Volume after 1 uL taken for Qubit (ul)
NAR_3	Reconcentrated DNA in 30 uL	Extraction 1 E2	12	41
NAR_5	Reconcentrated DNA in 30 uL	Extraction 2 E1	2.5	31.5
NAR_6	Reconcentrated DNA in 30 uL	Extraction 1 E1	2.7	31.7
NAR_7	Reconcentrated DNA in 30 uL	Extraction 2 E1	3	32
GHP_1	Reconcentrated DNA in 30 uL	Extraction 1 E1	3.75	32.75
GHP_2	Reconcentrated DNA in 30 uL	Extraction 3 E2	20	49
GHP_3	Reconcentrated DNA in 30 uL	Extraction 1 E2	21	50

• Added volume of Type 2 (column 4) to Type 1 tube (column 2)
  • Vortex and spin down

Qubit dsDNA BR assay

• Followed Qubit protocol for BR DNA

Sample	Avg ng/μl
Std 1	170 RFU
Std 2	20487 RFU
NAR_3	12.7
NAR_5	20.0
NAR_6	20.6
NAR_7	21.6
GHP_1	16.3
GHP_2	7.78
GHP_3	6.82
MCD_10	Too Low

Confirming that I have enough DNA in these samples:

Sample	Qubit concentration (ng/uL)	Vol. of Tris (uL)	Total DNA (ng)
NAR_3	12.7	41	520.7
NAR_5	20.0	31.5	630
NAR_6	20.6	31.7	653.02
NAR_7	21.6	32	691.2
GHP_1	16.3	32.75	533.83
GHP_2	7.78	49	381.22
GHP_3	6.82	50	341
MCD_10	Too Low	46	Too Low

GHP_2 and GHP_3 are too low, so adding more DNA:

Sample	Type to add	Qubit conc. (ng/uL)	Vol. of sample to add (uL)	Total volume (uL)
GHP_2	Extraction 3 E1	144	2	51
GHP_3	Extraction 1 E1	180	1	51

The MCD_10 sample DNA concentration is too low, so I will proceed using DNA from Extraction 2 E1 for this sample. Ready to move on to sonication.