ReRun5
- Gregg Randolph
MasterMix (make for 16 plates in 50mL conical tube)
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 2200 | 6600 |
0.45 | 10M dNTPs | 2200 | 990 |
0.3 | Kapa HiFi HotStart DNA Pol | 2200 | 660 |
5.25 | HPLC H2O | 2200 | 15950 |
9 | Total Volume | 2200 | 24200 |
Add 9 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “NS5 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plate | 16S Primer | ITS Primer |
|---|---|---|
HW1 | 16S0A1 | ITS0A1 |
16S0B1 | ITS0B1 | |
HW2 | 16S0C1 | ITS0C1 |
16S0D1 | ITS0D1 | |
NW1 | 16S0E1 | ITS0E1 |
16S0F1 | ITS0F1 | |
NW2 | 16S0G1 | ITS0G1 |
16S0H1 | ITS0H1 | |
NW3 | 16S0A2 | ITS0A2 |
16S0B2 | ITS0B2 | |
NW4 | 16S0C2 | ITS0C2 |
16S0D2 | ITS0D2 | |
BW1 | 16S0E2 | ITS0E2 |
16S0F2 | ITS0F2 | |
MO1 | 16S0G2 | ITS0G2 |
16S0H2 | ITS0H2 | |
MO2 | 16S0A3 | ITS0A3 |
16S0B3 | ITS0B3 | |
ROCK1 | 16S0C3 | ITS0C3 |
16S0D3 | ITS0D3 | |
LVD1 | 16S0A11 |
|
16S0B11 |
| |
LVD2 | 16S0E11 |
|
16S0F11 |
| |
LVD3 | 16S0G12 |
|
16S0H12 |
| |
LVD4 | 16S0C4 |
|
16S0D4 |
| |
TT6 | 16S0G4 |
|
16S0H4 |
| |
TT7 | 16S0E3 |
|
16S0F3 |
| |
MO3 | 16S0E4 |
|
16S0F4 |
| |
AH1 | 16S0G3 |
|
16S0H3 |
| |
AH2 | 16S0A4 |
|
16S0B4 |
|
qPCR and Sequencing
16S
Pool concentration = 2.87 nM
1nM Dilution = 69.6 ul Pool + 130.4 ul RSB
Load = 90 ul 1 nM + 9 ul PhiX + 1 ul RSB