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Well Position | Sample |
|---|---|
A01 | |
B01 | |
C01 | |
D01 | |
E01 | |
F01 | |
G01 | |
H01 | |
A02 | |
B02 | |
C02 | |
D02 | |
E02 | |
F02 | |
G02 | |
H02 | |
A03 | |
B03 | |
C03 | |
D03 | |
E03 | |
F03 | |
G03 | |
H03 | |
A04 | |
B04 | |
C04 | |
D04 | |
E04 | |
F04 | |
G04 | |
H04 | |
A05 | |
B05 | |
C05 | |
D05 | |
E05 | |
F05 | |
G05 | |
H05 | |
A06 | |
B06 | |
C06 | |
D06 | |
E06 | |
F06 | |
G06 | |
H06 | |
A07 | |
B07 | |
C07 | |
D07 | |
E07 | |
F07 | Mock Community |
G07 | Mock Community |
H07 | Mock Community |
PCR1 (Manual):
Create MasterMix:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 224 | 840 |
0.45 | 10M dNTPs | 224 | 126 |
0.3 | Kapa HiFi HotStart DNA Pol | 224 | 84 |
3.25 | HPLC H2O | 224 | 910 |
7 | Total Volume | 224 | 1960 |
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Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00 |
98 | 15X | 0:30 |
62 | 15X | 0:30 |
72 | 15X | 0:30 |
72 | 1X | 5:00 |
4 | 1X | 0:00 |
PCR2 Mastermix Preparation:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HF Buffer | 112 | 540 |
0.45 | 10M dNTPs | 112 | 81 |
0.3 | Kapa HiFi HotStart DNA Pol | 112 | 54 |
0.5 ul | 5 uM F and R FlowCell Primers | 112 | 90 |
0.75 | HPLC H2O | 112 | 135 |
5 | Total Volume | 112 | 900 |
Add 5 ul master mix to all wells of 4 hard shell full skirted plates. Seal with tape seals and store in refrigerator.
Intermediate Cleanup:
The majority of this was done on the Nimbus platform using “AxyPrep MagBead PCR1 No MM”
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*PCR2 actually had 20 cycles, not 19
Final Cleanup:
Equilibrate Beads to room Temperature
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