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| Info |
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Data was returned on 9-21-2021. Data was transferred to client and invoiced. M01247 |
5CM1 (16S only) | 5CM2 (16S only) | 5CM3 (16S only) | 5CM4 (16S only) |
|---|---|---|---|
MasterMix (make for 8 plates in 15mL conical tube)
ul/rxn | Reagent | # of rxns | ul needed (1.5X) |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 768 | 3456 |
0.45 | 10M dNTPs | 768 | 518.4 |
0.3 | Kapa HiFi HotStart DNA Pol | 768 | 345.6 |
7.25 | HPLC H2O | 768 | 8352 |
11 | Total Volume | 768 | 12672 |
*Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “NovaSeq4 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plate | 16S MID | ITS MID |
|---|---|---|
5CM1 | 16S0A1 | N/A |
16S0B1 | N/A | |
5CM2 | 16S0C1 | N/A |
16S0D1 | N/A | |
5CM3 | 16S0E1 | N/A |
16S0F1 | N/A | |
5CM4 | 16S0G1 | N/A |
16S0H1 | N/A |
Run on Thermocycler Program GSAF36:
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:
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 NovaSeq5_Plate1
Make 1:1000 dilutions of column 3 from the PCR plates 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 | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 |
| NTC | NTC | NTC | ||||
B | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 |
| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | ||||
C | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | ||||
D | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 |
| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | ||||
E | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | ||||
F | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 |
| 2 pM Std | 2 pM Std | 2 pM Std | ||||
G | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 |
| 20 pM Std | 20 pM Std | 20 pM Std | ||||
H | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 |
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Make a 1:1000 dilution of HMax_PP Final Pool to check quality of pippin elution before sending out for RFS.
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) | 60 | 600 |
2 ul | Primer Premix (10X) | 60 | 120 |
4 ul | Ultra Pure Water | 60 | 240 |
16 ul | Total Volume | 60 | 960 |
Add 4 ul of template, pool, or standards to each well:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 | HMax_PP_Final |
| NTC | NTC | NTC | |||
B | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 | 5RM1_1nm_Pool |
| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | |||
C | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | ||||
D | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 |
| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | ||||
E | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | ||||
F | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 |
| 2 pM Std | 2 pM Std | 2 pM Std | ||||
G | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 |
| 20 pM Std | 20 pM Std | 20 pM Std | ||||
H | 5CM1_16S_Col3 | 5CM2_16S_Col3 | 5CM3_16S_Col3 | 5CM4_16S_Col3 |
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Results:
Average results for the following plates (column 3):
5CM1_16S: 6.03 nanomoles
5CM2_16S: 9.32 nanomoles
5CM3_16S: 3.52 nanomoles
5CM4_16S: 9.77 nanomoles
Results for HMax_PP_Final can be viewed in result report below and have been added to the HMax page here.
Full result report can be viewed below:
| View file | ||
|---|---|---|
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Pooling and Shipping:
The client, Chris MacGlover, is concerned about the nasal swabs not sequencing as occurred with his last attempt. We will send 3 nasal, one excontrol, and one other sample to be fragment analyzed at CU:
5CM1 A4 (18-143N), 5CM1 H12 (SB33N), 5CM2 A4 ( 18-143T), 5CM3 H12 (51N), and 5CM1 A3 (excontrol)
Fragment Analysis returned from the Genomics Core lead us to allow sequencing to proceed.
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Well | Conc pg/ul | Sample Description |
|---|---|---|
EL1 | 2350 | Electronic Ladder |
A1 | 2380 | 18-143T |
B1 | 1090 | SB33N |
C1 | 1390 | 18-143N |
D1 | 1450 | 51N |
E1 | 688 | excontrol |
F1 | 1890 | 5CM pool |