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.
*DO NOT USE THE FOLLOWING 2 STEP PCR MID PLATES (16S OR ITS) FOR 4AH1: A2, B2, A4, B4, C8, D8
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; 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 ul H2O; 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 to clean transparent PCR plate.
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.