Illumina DNA Prep
Measure DNA concentration via Synergy HTX
If samples are above 16 ng/ul, normalize that sample between 4 and 16 ng/ul.
If more than 5 samples per plate, use Nimbus to normalize to 10 ng/ul
Add 30 ul of each sample to hard shell 96 well plate
Bring BLT and TB1 to RT
Add 20 ul Tagmentation Master Mix after vortexing:
ul per Rxn | Reagent | Thaw notes | Rxns | ul Needed |
|---|---|---|---|---|
11 | BLT (Vortex 10s to resuspend!!) | Vortex to mix, no centrifuge | ||
11 | TB1 |
Mix via pipetting 10x
Seal, quick spin, and run on cycler program TAG:
Step | Temp | Time (mm:ss) |
|---|---|---|
Preheat | 55 C | infinity |
Tagmentation | 55 C | 15:00 |
Cool | 10 C | infinity |
Heat TSB at 37 C for 10 minutes if precipitate observed
Spin down tagmentation plate
Add 10 ul TSB to each well, mix by pipette 10x
Seal, quick spin, and run on Cycler Program:
Step | Temp | Time (mm:ss) |
|---|---|---|
Preheat | 37 C | infinity |
Stop Tagment | 37 C | 15:00 |
Cool | 10 C | infinity |
Place plate on magnetic stand and allow to clear 3 minutes
Remove and discard supernatant
Remove plate from magnets
Slowly add 100 ul TWB directly to beads, pipette slowly until resuspended
Place plate on magnetic stand and allow to clear 3 minutes
Remove and discard supernatant
Remove plate from magnets
Slowly add 100 ul TWB directly to beads, pipette slowly until resuspended
Place plate on magnetic stand and allow to clear at least 3 minutes, leave until ready next MM is ready to add:
ul per Rxn | Reagent | Thaw | Rxns | ul Needed |
|---|---|---|---|---|
22 | EPM | On ice, invert to mix, quick spin | ||
22 | H2O | At RT, vortex, quick spin |
Remove Supernatant
Remove fro magnet
Add 40 ul MM from above and pipette to resuspend
Seal and quick spin
10 ul pre paired i7 and i5 index adapters
Pipette 40 ul up and down to mix
Run on Cycler Program:
Temp | Time (mm:ss) | Cycles |
|---|---|---|
68 C | 3:00 | 1 |
98 C | 3:00 | 1 |
98 C | 0:45 | 5 |
62 C | 00:30 | |
68 C | 2:00 | |
68 C | 1:00 | 1 |
10 C | 0:00 | 1 |
Cleanup:
Equilibrate Sample Purification Beads to RT for 30 minutes and invert to resuspend
Place PCR plate on magnetic stand and wait 5 minutes
Transfer 45 ul to deepwell or midi plate
Add 45 ul SPB to each well, pipette up and down 10x
Incubate at RT for 5 min
Place on magnet for 5 minutes
Add 15 ul SPB to new midi plate
Transfer 125 ul supernatant to new midi plate from above and mix 10x
Discard 1st plate
Incubate at RT for 5 minutes
Place on magnet for 5 minutes
Remove and discard supernatant, without moving plate
Add 200 ul EtOH
Incubate 30s
Remove 200 ul
Add 200 ul EtOH
Incubate 30s
Remove 200 ul
Remove Residual EtOH
Air Dry for 5 minutes
Remove from magnet
Add 32 ul RSB and pipette to resuspend
Incubate at RT 2 minutes
Return to magnet 2 minutes, Transfer 30 ul to new pcr plate.
Spot Check preps via qPCR and/or bioanalyzer
MyBaits:
Pool 4+ libraries together in equal volumes