Test samples were previously extracted.
Make sample spreadsheet and plate test samples by adding 30uL of sample to the corresponding well.
Quantify samples on Synergy HTX.
Normalize samples to 10ng/uL.
Choose 4 plant and 4 stool to run PCR test.
Make 1 step PCR MMX and plate in the template format below (Replace P1, P2, …. and S1, S2… with actual sample names):
PCR MasterMix
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 80 | 240 |
0.45 | 10M dNTPs | 80 | 36 |
0.3 | Kapa HiFi HotStart DNA Pol | 80 | 24 |
7.25 | HPLC H2O | 80 | 580 |
11 | Total Volume | 80 | 880 |
Add 11 ul to each well of a hard shell, full skirt plate. Add 2 uL of primers and 2uL of template to each well.
Primers: TRNL01
Template Format:
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) | ||||
B | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) | ||||
C | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) | ||||
D | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) | ||||
E | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) | ||||
F | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) | ||||
G | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) | ||||
H | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) |
Run on thermocycler program TRNL_T*:
Step | Temp C | Cycles | Time |
|---|---|---|---|
Denature | 95 | 1X | 10:00 |
Annealing | 95 | 35X | 0:30 |
Annealing** (Row E) | 50 | 35X | 0:30 |
Extension/Elongation | 72 | 35X | 2:00 |
Hold | 4 | 1X | 0:00 |
*TRNL_T program will test the efficiency of the PCR/annealing temperature by running it on a temperature gradient for each row. The table below shows the annealing temperature for each row.
**
Row | Annealing Temperature |
|---|---|
A | 60 |
B | 57 |
C | 55 |
D | 53 |
E | 50 |
F | 48 |
G | 46 |
H | 44 |
MagBead Cleanup:
Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”
Manually, it was done:
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)
qPCR TRNL_Test
Make 1:1000 dilutions of column 1-8 from the PCR plate by adding 1 ul to 999 ul TE in a deep well plate:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) |
| NTC | NTC | NTC |
B | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) |
| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) |
| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) |
| 2 pM Std | 2 pM Std | 2 pM Std |
G | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) |
| 20 pM Std | 20 pM Std | 20 pM Std |
H | P2 (B1) | P7 (G1) | P9 (A2) | P14 (G2) | GM2 (C3) | SB4 (H3) | SB1 (D4) | AT16 (F4) |
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Add 16 ul of Illumina Library Quantification MasterMix to each well:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
10 ul | KAPA SYBR FAST qPCR MM (2X) | 110 | 1100 |
2 ul | Primer Premix (10X) | 110 | 220 |
4 ul | Ultra Pure Water | 110 | 440 |
16 ul | Total Volume | 110 | 1760 |
Add 4 ul of template, pool, or standards to each well:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | P2_A | P7_A | P9_A | P14_A | GM2_A | SB4_A | SB1_A | AT16_A |
| NTC | NTC | NTC |
B | P2_B | P7_B | P9_B | P14_B | GM2_B | SB4_B | SB1_B | AT16_B |
| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C | P2_C | P7_C | P9_C | P14_C | GM2_C | SB4_C | SB1_C | AT16_C |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D | P2_D | P7_D | P9_D | P14_D | GM2_D | SB4_D | SB1_D | AT16_D |
| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E | P2_E | P7_E | P9_E | P14_E | GM2_E | SB4_E | SB1_E | AT16_E |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F | P2_F | P7_F | P9_F | P14_F | GM2_F | SB4_F | SB1_F | AT16_F |
| 2 pM Std | 2 pM Std | 2 pM Std |
G | P2_G | P7_G | P9_G | P14_G | GM2_G | SB4_G | SB1_G | AT16_G |
| 20 pM Std | 20 pM Std | 20 pM Std |
H | P2_H | P7_H | P9_H | P14_H | GM2_H | SB4_H | SB1_H | AT16_H |
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Results:
Average results