DNA Library Prep Test on 4 Sea Stars With 2 Min Sonication with 500ng, 100ng Going into DNA Prep
Using 1/4th reactions of the KAPA HyperPrep DNA Library Prep Kit
Sonication
- 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 |
| CPAB 007 |
36 |
14.7 |
65.3 |
| CPAB 003 |
50 |
8.1 |
71.9 |
| BPH1 |
56 |
10.9 |
69.1 |
| CHSB 004 |
66 |
12.2 |
67.8 |
- Ran sonicator for 2 minutes at 25% amplitude, 15 seconds on 15 seconds off. Samples were spun down after 1 minute (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
End Repair and A-tailing
- 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)
Adapter Ligation
- 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
| Sample |
LMM |
Adapter |
| 36 |
11.25μl |
1.25μl 12 |
| 50 |
11.25μl |
1.25μl 2 |
| 56 |
11.25μl |
1.25μl 8 |
| 66 |
11.25μl |
1.25μl 6 |
- Pipetted all samples up and down with the multichannel and spun down
- Incubated on the shaker at room temp for 1 hour 200rpm
0.8X Cleanup
- 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
Library Amplification
- Did not make master mix for 4 samples
- Each tube from the cleanup got 6.25ul KAPA HiFi HotStart Ready Mix
- Each sample got 0.75ul of each index primer (see primers below)
| Sample |
Index pair (0.75ul each) |
| 36 |
504 704 |
| 50 |
506 706 |
| 56 |
506 706 |
| 66 |
507 707 |
- Vortexed and spun down samples
- Placed samples in the thermocycler Genomic PCR program for 6 cycles
1X Cleanup
- 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 21μl 10mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
- Placed tubes back onto the magnet stand and removed 20ul supernatant when clear to new labeled PCR strip tubes
QC
| Sample |
Std 1 |
Std 2 |
Avg ng/μl |
| 36 |
180 RFU |
22462 RFU |
19.3 |
| 50 |
- |
- |
25.6 |
| 56 |
- |
- |
21.2 |
| 66 |
- |
- |
8.7 |
- Ran the D1000 TapeStation