2nd DNA Library Prep Test 4 Sea Stars

DNA Library Prep Test on 4 Sea Stars With 2.5 Min Sonication with 500ng, 100ng Going into DNA Prep

Prepared 500ng of each sample in 80ul of 10mM tris HCL. Samples were chosen to not take too much DNA so that if this didn’t work, I could take 500ng again from them

Sample	Library number	ul for 500ng	ul tris to 80ul
CPDB 009	29	10.3	69.7
CPBP 009	43	10.8	69.2
CPBO 006	62	9.7	70.3
CPOI 008	71	13.6	66.4

Ran sonicator for 2.5 minutes at 25% amplitude, 15 seconds on 15 seconds off. Samples were spun down after 1.25 minutes (general sonicator protocol)
1X bead clean after sonication, followed protocol
Samples were re-suspended in 62.5ul 10mM tris HCl, this is 5*12.5, which is the input volume to the kit. I was then able to take 12.5ul from each sonicated sample and that should be ~100ng

Prepared end repair and a-tailing master mix on ice:
- ERAT buffer 1.75μl * 4.4 = 7.7μl
- ERAT enzyme 0.75μl * 4.4 = 3.3μl
Made 4 PCR strip tubes each with 12.5μl of 100ng sheared DNA
Added 2.5μl of ERAT master mix to each sample
Vortexed and spun down
Placed samples in thermocyler A-tailing program in JONP login (~ 1 hour)

Prepared ligation master mix on ice (no vortexing of the ligase):
- ligation buffer 7.5μl * 4.2 = 31.5μl
- DNA ligase 2.5μl * 4.2 = 10.5μl
- nuclease free water 1.25μl * 4.2 = 5.25μl
Added ligation master mix and appropriate planned adapters to each sample. Adapters were added last to minimize adapter-adapter ligation

note, for #29, 1.25ul of 40uM adapter was added, mistake was noticed and 1.25ul of 15uM adapter was used for other samples
Pipetted all samples up and down with the multichannel and spun down
Incubated on the shaker at room temp for 1 hour 200rpm

Made fresh 80% EtOH
Took KAPA Pure Beads out of fridge beforehand to warm to room temp
After incubation at RT, added 22μl of KAPA pure beads to each sample and pipetted up and down at least 10 times to mix beads careful to avoid bubbles
Placed tubes on shaker at room temp for 15 minutes
Placed tubes on magnet plate and removed supernatant from tubes when it was fully clear not disturbing the beads
Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
Removed supernatant from each tube on the magnet plate without disturbing the beads
Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
Removed ALL the supernatant from each tube on the magnet plate without disturbing the beads. Extra EtOH blobs were removed with p20 pipette tips
Resuspended beads in 6μl 10mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
Placed tubes back onto the magnet stand and removed 5ul supernatant when clear to new labeled PCR strip tubes

After PCR, added 12.5μl of KAPA pure beads to each sample and pipetted up and down at least 10 times to mix beads careful to avoid bubbles
Placed tubes on shaker at room temp for 15 minutes
Placed tubes on magnet plate and removed supernatant from tubes when it was fully clear not disturbing the beads
Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
Removed supernatant from each tube on the magnet plate without disturbing the beads
Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
Removed ALL the supernatant from each tube on the magnet plate without disturbing the beads. Extra EtOH blobs were removed with p20 pipette tips
Resuspended beads in 17μl 10mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
Placed tubes back onto the magnet stand and removed 16ul supernatant when clear to new labeled PCR strip tubes

Sample	Std 1	Std 2	Avg ng/μl
29	176 RFU	21321 RFU	57.4
43	-	-	39.8
62	-	-	49.6
71	-	-	53.3

Written on July 6, 2021