We will be comparing the difference in results for 4AH1 using the 2 step PCR and the 1 step PCR. The 1 step PCR protocol used for 4AH1 is under the page named “NovaSeq4 1Step PCR and qPCR”. Below is the protocol used for the 2 step PCR. We will also be repeating the 2 step for PCR for GC2017-2.
*DO NOT USE THE FOLLOWING 2 STEP PCR MID PLATES (16S OR ITS) FOR 4AH1: A2, B2, A4, B4, C8, D8
4AH1 PCR1 MasterMix (make for 4 plates in 5mL tube)
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 500 | 1500 |
0.45 | 10M dNTPs | 500 | 225 |
0.3 | Kapa HiFi HotStart DNA Pol | 500 | 150 |
3.25 | HPLC H2O | 500 | 1625 |
7 | Total Volume | 500 | 3500 |
...
Store at 4C when cycler reaches below 40C unless immediately proceeding to next step.
Cleanup of PCR 1 product
Equilibrate Beads to room Temperature
Add 24 ul of MagBeads to each well; well and 15 ul of replicate to same well of replicate
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 65 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
Reaspirate from each well to assure maximum EtOH removal
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 30 40 ul H2OTE; pipette mix 10+ times. Incubate 2 minutes at RT.
Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Transfer 30uL 40 ul to clean labeled transparent PCR plate. plate (Plate name_PCR_MIDs)
4AH2 PCR2 MasterMix (make for 2 plates in 1.5mL tube)
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Phusion HF Buffer | 300 | 900 |
0.45 | 10M dNTPs | 300 | 135 |
0.3 | Kapa HiFi HotStart DNA Pol | 300 | 90 |
0.5 ul | 5 uM F and R FlowCell Primers | 300 | 150 |
0.75 | HPLC H2O | 300 | 225 |
5 | Total Volume | 300 | 1500 |
...
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00 |
98 | 20X | 0:30 |
55* | 20X | 0:30 |
72 | 20X | 0:30 |
72 | 1X | 5:00 |
4 | 1X | 0:00 |
Final Cleanup:
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 40 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 40 ul to a clean transparent PCR plate.
Run on qPCR:
Column 3 of 4AH1_16S and 4AH1_ITS was used for quantification.
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GC2017-2 PCR1 MasterMix (make for 4 plates in 5mL tube)
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 500 | 1500 |
0.45 | 10M dNTPs | 500 | 225 |
0.3 | Kapa HiFi HotStart DNA Pol | 500 | 150 |
3.25 | HPLC H2O | 500 | 1625 |
7 | Total Volume | 500 | 3500 |
Add 7 ul to each well of 16 hard shell, full skirt plate. Add 2 ul of template and 6 ul of the primers.
Spin down resulting “PCR1”s and then add to thermocyclers running “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 |
Store at 4C when cycler reaches below 40C unless immediately proceeding to next step.
Cleanup of PCR 1 product
Equilibrate Beads to room Temperature
Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate
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 65 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
Reaspirate from each well to assure maximum EtOH removal
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.
Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Transfer 40 ul to labeled transparent plate (Plate name_PCR_MIDs)
GC2017-2 PCR2 MasterMix (make for 2 plates in 1.5mL tube)
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Phusion HF Buffer | 300 | 900 |
0.45 | 10M dNTPs | 300 | 135 |
0.3 | Kapa HiFi HotStart DNA Pol | 300 | 90 |
0.5 ul | 5 uM F and R FlowCell Primers | 300 | 150 |
0.75 | HPLC H2O | 300 | 225 |
5 | Total Volume | 300 | 1500 |
Add 5 ul master mix to all wells of 2 hard shell full skirted plates. Seal with tape seals and store in refrigerator if not using immediately. Add 10ul of template from PCR1. Run PCR2 on 35GSAF2 program.
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00 |
98 | 20X | 0:30 |
55* | 20X | 0:30 |
72 | 20X | 0:30 |
72 | 1X | 5:00 |
4 | 1X | 0:00 |
Final Cleanup:
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 40 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 40 ul to a clean transparent PCR plate.
Run on qPCR:
Column 3 of GC2017-2_16S and GC2017-2_ITS was used for quantification.
GC2017-2_16S:
GC2017-2_ITS: