SeqWell WGS Testing
Tested with Lambda DNA at 10 ng/ul adding 5 ul of it and 5 ul TE for a total of 50 ng
Setup
Thaw 5X Rxn Buffer (orange), Tagmentation Enhancer(yellow), Diluent(red), Read 2 Adapter(blue), UDI primer plate, and MAGwise beads at RT
Place Tnx Read 1 Tagging Reagent(white), Ligase(green), and Amplification Master Mix(clear) on ice
Prepare 80% EtOH, 500 ul per sample
Set out 10 mM Tris-HCl and low EDTA TE
Tagmentation
Vortex TnX Read 1 at 60% vortex speed, Vortex yellow and orange tubes at 90% speed.
Reagent | ul/rxn | Rxns | ul needed |
|---|---|---|---|
TnX Read 1(white) | 8 | 1 | 8 |
Tagmentation Enhancer(yellow) | 2 | 1 | 2 |
5X Rxn Buffer(orange) | 5 | 1 | 5 |
Vortex Tag MM at 70% speed
Add 5 ul TE and 5 ul Lambda DNA at 10 ng/ul for a total of 50 ng
Pipette mix 10+ times at 15ul then quick spin
Run on Thermocycler program XTAG:
37C | 7:00 |
95C | 3:00 |
4C | hold |
Return Tagmentation Enhancer(yellow), TnX Read 1 Tagging Reagent(white), Diluent(red) to the freezer. Keep 5X rxn Buffer on ice. Proceed Immediately To Ligation
Ligation
Prepare MM while XTAG is running. Vortex at 70% speed the green, orange, and blue tubes:
Reagent | ul/rxn | Rxns | ul needed |
|---|---|---|---|
Read 2 Adapter(blue) | 4 | 1.1 | 4.4 |
5X Rxn Buffer (orange) | 3 | 1.1 | 3.3 |
Ultrapure Water | 7 | 1.1 | 7.7 |
Ligase (green) | 1 | 1.1 | 1.1 |
Vortex Ligation MM at 70% speed. Place on ice until XTAG program concludes.
Spin down Tagmentation plate and add 15 ul MM per rxn
Pipette mix 10+x at 15ul. Quick spin and add to thermocycler LIGATE program:
25C | 5:00 |
4C | hold |
Once program completes, proceed immediately to 1st bead cleanup
Post Ligation Bead Clean
Spin down rxn plate
Vortex RT MAGwise beads and add 40 ul (1X) to each rxn well
Incubate 5 Min
Move to Magnet and incubate 5 min on it
Remove and discard supernatant
Add 150 ul 80% EtOH, Incubate 30s, Remove 150ul EtOH
Add 150 ul 80% EtOH, Incubate 30s, Remove 150ul EtOH
Remove from magnet, Spin down, Return to magnet for <30s, Remove remaining EtOH
Remove from Magnet, Add 22ul 10 mM Tris-HCl to each well and resuspend
Incubate 5 Min
Return to magnet and incubate 2 Min, Transfer 20 ul to new plate or well
Freeze at -20C or proceed to amplification
PCR amplification
Remove UDI indexing primers from freezer and thaw at RT
Remove 2X Amp Master Mix and thaw on ice, Vortex ar 70% speed
Vortex and spin down the UDI primers
Pipette mix primers 5x at 5 ul, Then transfer 5 ul to each ligation rxn well
Add 25 ul of well mixed 2X Amp MM to each well
Run PCR Amplification on a thermocycler:
Temp | Time | Cycles |
|---|---|---|
98C | 0:45 | 1 |
98C | 0:15 |
5 |
60C | 0:30 | |
72C | 0:30 | |
72C | 1:00 | 1 |
4C | hold | 1 |
Proceed Immediately to Post-PCR cleanup or freeze at -20C
Post PCR Bead Clean
Add 50 ul Ultrapure Water and Pipette mix 10-15x at 50 ul
Add 70 ul MAGwise beads to each well, Pipette mix 15x at 70 ul
Incubate 5 Minutes
Place on Magnet and incubate 5 minutes
Remove and discard supernatant
Add 200 ul 80% EtOH, Incubate 30s, Remove EtOH
Add 200 ul 80% EtOH, Incubate 30s, Remove EtOH
Spin down, Return to magnet for < 30s
Remove remaining EtOH with 20 ul tip
Remove from magnet and resuspend with 26 ul low E TE
Incubate 5 minutes
Return to magnet and incubate 2 minutes
Transfer 24 ul to new well or plate
TapeStation qPCR
Run 10x diluted Run products on HD5000 tapestation
Run on qPCR