This experiment is to verify fullITS primers can be sequenced. Primer Source Paper
Primer bases:
ITS-p5 (forward): CCTTATCAYTTAGAGGAAGGAG
ITS-p4 (reverse): CCGCTTAKTGATATGCTTAAA
94 °C for 4 min, followed by 34 cycles of 30 s at 94 °C, 40 s at 55 °C (or 58 °C) and 1 min at 72 °C, with a final step of 10 min at 72 °C.
Dilute and Array Primers
Primers arrived 100uM in 50 ul
Add 575 to get primers to 8 uM with Integra Pipette using Pipette/Mix setting
Add 87.5 ul to new Primer plates
Use Nimbus to array first four plates using FULLITS_Primer_Prep_Adjustable
PCR MasterMix
ul/rxn | Reagent | # of rxns | ul needed |
|---|
3
5X Kapa HiFi Buffer
384
1320
0.45
10M dNTPs
384
198
0.3
7.5 | Kapa HiFi HotStart DNA |
2X |
120 |
900 |
4. |
5 | HPLC H2O |
120 |
540 |
12 | Total Volume | 384 |
1440 |
Add 11 ul to each well of a hard shell, full skirt plate. Add 2 1 uL of 0.5 uM primers and 2uL of template to each well.
Primers:
Run TRNL fullITS plates on thermocycler program TRNL35fullITS:
Step | Temp C | Cycles | Time |
|---|---|---|---|
Denature | 95 | 1X | 10:00 |
Denature |
94 | 35X | 0:30 | |
Annealing** (Row C) | 55 | 35X | 0: |
40 | ||
Extension/Elongation | 72 | 35X |
1: |
00 | ||
Final Extension | 72 | 1X |
10:00 | |||
Hold | 4 | 1X | 0:00 |
Run 16S plates on Thermocycler Program GSAF35:
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00* |
98 | 35X | 0:30 |
62 | 35X | 0:30 |
72 | 35X | 0:30 |
72 | 1X | 5:00 |
4 | 1X | 0:00 |
Pool duplicates together.
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Pool 2 ul of the TRNL and 16S samples separately. Make 1:1000 dilutions of each pool and run in triplicate.
Make 1:1000 dilutions of columns 1,6, and 12 for each plate using 999 of TE and 1uL of sample into a deep well plate.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 |
| NTC | NTC | NTC | ||
B | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 |
| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | ||
C | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | ||
D | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 |
| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | ||
E | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | ||
F | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 |
| 2 pM Std | 2 pM Std | 2 pM Std | ||
G | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 |
| 20 pM Std | 20 pM Std | 20 pM Std | ||
H | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 |
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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 | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 | TRNL1_1_16S_Pool |
| NTC | NTC | NTC | |
B | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 | TRNL1_1_16S_Pool |
| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | |
C | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 | TRNL1_1_16S_Pool |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | |
D | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 | TRNL1_1_T_Pool |
| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | |
E | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 | TRNL1_1_T_Pool |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | |
F | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 | TRNL1_1_T_Pool |
| 2 pM Std | 2 pM Std | 2 pM Std | |
G | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 |
| 20 pM Std | 20 pM Std | 20 pM Std | ||
H | TRNL1_1_16S_Col1 | TRNL1_1_16S_Col6 | TRNL1_1_16S_Col12 | TRNL1_1_T_Col1 | TRNL1_1_T_Col6 | TRNL1_1_T_Col12 |
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Results:
The TRNL pool via standard size estimation returned a mean of ? nM. This should be adjusted for the difference between the standards' fragment sizes and the expected product size (452 vs 220). 9.41x(452/220) = 19.33 nM
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