We will run 6 samples of TRNL (3 soil and 3 plant) as well as 6 samples of Bo Steven’s AMF samples at 50 cycles on the BioAnalyzer to make sure the PCR is optimized to obtain a sufficient amount of product.
PCR MasterMix
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 15 | 45 |
0.45 | 10M dNTPs | 15 | 6.75 |
0.3 | Kapa HiFi HotStart DNA Pol | 15 | 4.5 |
7.25 | HPLC H2O | 15 | 108.75 |
11 | Total Volume | 15 | 165 |
Add 11 ul to 6 wells of two PCR strip tubes. Add 2 uL of primers and 2uL of template to each well.
TRNL Primers: A1 Position: TRNL02, TRNL07, TRNL11, TRNL14, TRNL19, TRNL23 (Strip Tube 1)
AMF Primers: AMF06 (Strip Tube 2)
Template Format:
| Strip Tube 1: TRNL | Strip Tube 2: AMF |
|---|---|---|
A | P2 (B1) (TRNL02: A1) | BS_PLT1 (B1) |
B | P9 (A2) (TRNL07: A1) | BS_PLT1 (E4) |
C | P14 (G2) (TRNL11: A1) | BS_PLT1 (H10) |
D | PB10 (E3) (TRNL14: A1) | BS_PLT2 (B1) |
E | GM1 (C4) (TRNL19: A1) | BS_PLT2 (E4) |
F | SS8 (E4) (TRNL23: A1) | BS_PLT2 (H10) |
G | ||
H |
Strip Tube 1: Run on thermocycler program TRNL_50:
Step | Temp C | Cycles | Time |
|---|---|---|---|
Denature | 95 | 1X | 10:00 |
Annealing | 95 | 50X | 0:30 |
Annealing | 50 | 50X | 0:30 |
Extension/Elongation | 72 | 50X | 2:00 |
Hold | 4 | 1X | 0:00 |
Strip Tube 2: Run on thermocycler program AMF_50:
Step | Temp C | Cycles | Time |
|---|---|---|---|
Denature | 95 | 1X | 10:00 |
Extension/Elongation | 74 | 1X | 9:00 |
Extension/Elongation | 72 | 50X | 1:00 |
Annealing | 94 | 50X | 0:30 |
Annealing | 55 | 50X | 0:30 |
Hold | 4 | 1X | 0:00 |
MagBead Cleanup:
Equilibrate Beads to room Temperature
Add 15uL of ultra pure water to each well.
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 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)
BioAnalyzer:
BioAnalyzer Chip Set up:
Well 1: TRNL_P2
Well 2: TRNL_P9
Well 3: TRNL_P14
Well4: TRNL_PB10
Well 5: TRNL_GM1
Well 6: TRNL_SS8
Well 7: BS_PLT1_B1
Well 8: BS_PLT1_E4
Well 9: BS_PLT1_H10
Well 10: BS_PLT2_B1
Well 11: BS_PLT2_E4
Well 12: BS_PLT2_H10