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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: _______________ (fill in when primers arrive).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”
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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