Quantify DNAs on Synergy HTX Plate Reader:
Normalize DNAs on Nimbus:
Had to define these semi skirted plates for the Nimbus. We might want to standardize to full skirted plates. They are less variable.
16S Library Prep:
Swap out KG4 G9’s blank for Mock Community positive control
Add 12 ul Master mix to each well:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Phusion HF Buffer | 800 | 2400 |
0.45 | 10M dNTPs | 800 | 360 |
1 | 0.0001ng/ul 16S Synthgene_GR | 800 | 800 |
0.3 | Kapa HiFi HotStart DNA Pol | 800 | 240 |
2.25 | HPLC H2O | 800 | 1800 |
7 | Total Volume | 800 | 5600 |
Add 6 ul 0.25 uM paired primers ____________________
Add 2 ul of appropriate gDNA to each well
Run on Thermocycler Program 35GSAF1:
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00 |
98 | 15X | 0:30 |
62 | 15X | 0:30 |
72 | 15X | 0:30 |
72 | 1X | 5:00 |
4 | 1X | 0:00 |
Pool Duplicates
Purify samples using modified manual AxyPrep MagBead PCR Clean-Up (GSAF uses AmpPure XP):
AmpPure XP PCR cleanup and AxyPrep MagBead have the exact same stock protocol!
Equilibrate Beads to room Temperature
Add 24 ul of MagBeads to each well; Pipette mix up and down 10 times.
Incubate at RT for 5 minutes
Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)
Remove 54 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 15 ul TE per GSAF protocol; pipette mix 10+ times
Incubate at RT for 2 minutes
Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Prepare next MasterMix while sample plate is on the magnet plate.
Add 20 ul FlowCell MasterMix to new plate:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Phusion HF Buffer | 400 | 1200 |
0.45 | 10M dNTPs | 400 | 180 |
0.3 | Kapa HiFi HotStart DNA Pol | 400 | 120 |
0.5 ul | 10 uM F and R FlowCell Primers | 400 | 200 |
0.75 | HPLC H2O | 400 | 300 |
5 | Total Volume | 400 | 2000 |
Transfer 10 ul from plate on magnet plate to new strip tubes.
Run on Thermocycler Program 35GSAF2:
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00 |
98 | 19X | 0:30 |
55* | 19X | 0:30 |
72 | 19X | 0:30 |
72 | 1X | 5:00 |
4 | 1X | 0:00 |
Purify samples using GSAF’s second modified MagBead protocol:
Equilibrate Beads to room Temperature
Add 15 ul H2O to each sample
Add 24 ul (0.8 x 60 ul) of MagBeads to each well; Pipette mix up and down 10 times
Incubate at RT for 5 minutes
Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)
Remove 54 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 23 ul TE per GSAF protocol; pipette mix 10 times
Incubate at RT for 2 minutes
Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Transfer 20 ul to a clean PCR plate.
____________________________________________________________________________________________________________
ITS Library Prep:
Swap out KG4 G9’s blank for Mock Community positive control
Add 12 ul Master mix to each well:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Phusion HF Buffer | 800 | 2400 |
0.45 | 10M dNTPs | 800 | 360 |
1 | 0.0001ng/ul 16S Synthgene_GR | 800 | 800 |
0.3 | Kapa HiFi HotStart DNA Pol | 800 | 240 |
2.25 | HPLC H2O | 800 | 1800 |
7 | Total Volume | 800 | 5600 |
Add 6 ul 0.25 uM paired primers ____________________
Add 2 ul of appropriate gDNA to each well
Run on Thermocycler Program 35GSAF1:
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00 |
98 | 15X | 0:30 |
62 | 15X | 0:30 |
72 | 15X | 0:30 |
72 | 1X | 5:00 |
4 | 1X | 0:00 |
Pool Duplicates
Purify samples using modified manual AxyPrep MagBead PCR Clean-Up (GSAF uses AmpPure XP):
AmpPure XP PCR cleanup and AxyPrep MagBead have the exact same stock protocol!
Equilibrate Beads to room Temperature
Add 24 ul of MagBeads to each well; Pipette mix up and down 10 times.
Incubate at RT for 5 minutes
Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)
Remove 54 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 15 ul TE per GSAF protocol; pipette mix 10+ times
Incubate at RT for 2 minutes
Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Prepare next MasterMix while sample plate is on the magnet plate.
Add 20 ul FlowCell MasterMix to new plate:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Phusion HF Buffer | 400 | 1200 |
0.45 | 10M dNTPs | 400 | 180 |
0.3 | Kapa HiFi HotStart DNA Pol | 400 | 120 |
0.5 ul | 10 uM F and R FlowCell Primers | 400 | 200 |
0.75 | HPLC H2O | 400 | 300 |
5 | Total Volume | 400 | 2000 |
Transfer 10 ul from plate on magnet plate to new strip tubes.
Run on Thermocycler Program 35GSAF2:
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00 |
98 | 19X | 0:30 |
55* | 19X | 0:30 |
72 | 19X | 0:30 |
72 | 1X | 5:00 |
4 | 1X | 0:00 |
Purify samples using GSAF’s second modified MagBead protocol:
Equilibrate Beads to room Temperature
Add 15 ul H2O to each sample
Add 24 ul (0.8 x 60 ul) of MagBeads to each well; Pipette mix up and down 10 times
Incubate at RT for 5 minutes
Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)
Remove 54 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 23 ul TE per GSAF protocol; pipette mix 10 times
Incubate at RT for 2 minutes
Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Transfer 20 ul to a clean PCR plate.