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NOTE: Plate sequenced for 16S only but ITS was prepped up until MagBead. No MagBead clean up was completed on ITS but is available if client would like to sequence for ITS in the future.
MasterMix (make for 4 plates in 5 mL tube)
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 288 | 1230 |
0.45 | 10M dNTPs | 288 | 184.5 |
0.3 | Kapa HiFi HotStart DNA Pol | 288 | 123 |
7.25 | HPLC H2O | 288 | 2972.5 |
11 | Total Volume | 288 | 4510 |
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Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00* |
98 | 36X | 0:30 |
62 | 36X | 0:30 |
72 | 36X | 0:30 |
72 | 1X | 5:00 |
4 | 1X | 0:00 |
MagBead Cleanup (16S Only):
Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”
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Equilibrate Beads to room Temperature
Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate
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
Make 1:1000 dilutions of column 1, 4, and 8 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:
Pool 2uL from each well into a single pool. Make 1:1000 dilution of 5RM1 Pool and run in duplicate on qPCR.
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| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | 5RM1_16S_Col1 | 5RM1_16S_Col4 | 5RM1_16S_Col8 | 5RM1_16S_Pool |
| NTC | NTC | NTC | ||||
B | 5RM1_16S_Col1 | 5RM1_16S_Col4 | 5RM1_16S_Col8 | 5RM1_16S_Pool |
| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | ||||
C | 5RM1_16S_Col1 | 5RM1_16S_Col4 | 5RM1_16S_Col8 |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | |||||
D | 5RM1_16S_Col1 | 5RM1_16S_Col4 | 5RM1_16S_Col8 |
| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | |||||
E | 5RM1_16S_Col1 | 5RM1_16S_Col4 | 5RM1_16S_Col8 |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | |||||
F | 5RM1_16S_Col1 | 5RM1_16S_Col4 | 5RM1_16S_Col8 |
| 2 pM Std | 2 pM Std | 2 pM Std | |||||
G | 5RM1_16S_Col1 | 5RM1_16S_Col4 | 5RM1_16S_Col8 |
| 20 pM Std | 20 pM Std | 20 pM Std | |||||
H | 5RM1_16S_Col1 | 5RM1_16S_Col4 | 5RM1_16S_Col8 |
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Results:
Average results for the following plates:
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| View file | ||
|---|---|---|
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iSeq Run:
Dilute 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.
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A double check of the 1nM pool dilution created for sequencing revealed it was actually 4.98 nM. We will repeat sequencing run.
iSeq Run 2:
Dilute 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.
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