This experiment is to verify these CO1 primers can be sequencedtest the latest extraction method, QIAamp Fast DNA Stool Mini Kit using the larger volume protocol. Primer Source Paper
Primer bases:
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95 °C for 60 s, 45 °C for 90 s, 72 °C for 90 s; 28 cycles of: 94 °C for 60 s, 50 °C for 90 s, 72 °C for 60 s
PCR MasterMix
ul/rxn | Reagent | # of rxns | ul needed |
|---|
3 | 5X Kapa HiFi Buffer | 50 | 150 |
0.45 | 10M dNTPs | 50 | 22.5 |
0.3 | Kapa HiFi HotStart DNA |
Pol |
50 |
15 |
7. |
25 | HPLC H2O |
50 |
362.5 |
11 | Total Volume |
50 |
550 |
Add 11 ul to each well of a hard shell, full skirt plate. Add 2 uL of 0.5 uM primers and 2uL of template to each well.
Primers:
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Step | Temp C | Cycles | Time |
|---|---|---|---|
Denature | 95 | 1X | 10:00 |
Denature | 94 | 35X | 1:00 |
Annealing** (Row C) | 50 | 35X | 1:30 |
Extension/Elongation | 72 | 35X | 1:00 |
Final Extension | 72 | 1X | 10:00 |
Hold | 4 | 1X | 0:00 |
Pool duplicates together.
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)
qPCR
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.
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1
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2
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3
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4
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5
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6
...
7
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8
...
9
...
10
...
11
...
12
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A
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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TRNL1_1_T_Col12
...
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NTC
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NTC
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NTC
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B
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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TRNL1_1_T_Col12
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0.0002 pM Std
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0.0002 pM Std
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0.0002 pM Std
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C
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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TRNL1_1_T_Col12
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0.002 pM Std
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0.002 pM Std
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0.002 pM Std
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D
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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TRNL1_1_T_Col12
...
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0.02 pM Std
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0.02 pM Std
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0.02 pM Std
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E
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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TRNL1_1_T_Col12
...
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0.2 pM Std
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0.2 pM Std
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0.2 pM Std
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F
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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TRNL1_1_T_Col12
...
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2 pM Std
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2 pM Std
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2 pM Std
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G
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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TRNL1_1_T_Col12
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20 pM Std
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20 pM Std
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20 pM Std
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H
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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TRNL1_1_T_Col12
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Add 16 ul of Illumina Library Quantification MasterMix to each well:
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ul/rxn
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Reagent
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# of rxns
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ul needed
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10 ul
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KAPA SYBR FAST qPCR MM (2X)
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110
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1100
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2 ul
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Primer Premix (10X)
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110
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220
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4 ul
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Ultra Pure Water
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110
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440
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16 ul
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Total Volume
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110
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1760
Add 4 ul of template, pool, or standards to each well:
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1
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2
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3
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4
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5
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6
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7
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8
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9
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10
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11
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12
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A
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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TRNL1_1_T_Col12
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TRNL1_1_16S_Pool
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...
NTC
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NTC
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NTC
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B
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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TRNL1_1_T_Col12
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TRNL1_1_16S_Pool
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0.0002 pM Std
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0.0002 pM Std
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0.0002 pM Std
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C
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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TRNL1_1_T_Col12
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TRNL1_1_16S_Pool
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0.002 pM Std
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0.002 pM Std
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0.002 pM Std
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D
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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TRNL1_1_T_Col12
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TRNL1_1_T_Pool
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0.02 pM Std
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0.02 pM Std
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0.02 pM Std
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E
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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TRNL1_1_T_Col12
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TRNL1_1_T_Pool
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0.2 pM Std
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0.2 pM Std
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0.2 pM Std
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F
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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TRNL1_1_T_Col12
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TRNL1_1_T_Pool
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2 pM Std
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2 pM Std
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2 pM Std
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G
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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TRNL1_1_T_Col12
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20 pM Std
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20 pM Std
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20 pM Std
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H
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TRNL1_1_16S_Col1
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TRNL1_1_16S_Col6
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TRNL1_1_16S_Col12
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TRNL1_1_T_Col1
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TRNL1_1_T_Col6
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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
16S is close enough to the standards' fragment size that the standard estimation can be used. 16S mean is 46.34 nM.
iSeq Sequencing:
Dilute TRNL Pool to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.
1000/Results = ul of Pool to Add
1000/19.33 = 52uL of Pool to Add
1000 - ul of Pool to Add = ul of “10 mM Tris 8.5” to Add
1000- 52 = 948 uL of 10mM Tris 8.5 to Add
Pool 16S Pool to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.
1000/Results = ul of Pool to Add
1000/46.34 = 22 uL of Pool to Add
1000 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add
1000- 22 = 978 uL of 10mM Tris 8.5
Combine 100 ul TRNL 1 nM pool to 100 ul 16S 1 nM pool to create a combined 1 nM pool
Dilute 1 nM full pool to loading concentration of 50 pM:
Add 5 ul 1 nM Pool to 85 ul “10 mM Tris 8.5” and 10 ul 50 pM PhiX
Remove iSeq 100 i1 Flow Cell from refrigerator 5’s crisper drawer and open white foil pack and allow to equilibrate to RT for 10-15 minutes.
Open “iSeq 100 i1 Reagent Cartridge v2”. Turn on iSeq100
Click on “Sequence”. Watch Video. Do what video tells you to do. Follow on screen instructions until run starts.
Run samples on TapeStation with D1000 kit:
Positive Control Butterfly (D1):
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Extraction Blank(A2):
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Typical Amplification (42/D2):
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Worst Amplification (43/E2):
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Best Amplification (46/H2):
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