Plate | 16S Primer | ITS Primer | AMF | ||
|---|---|---|---|---|---|
6LVD5 | 16S0A4 | ITS0A4 | AMF01 | 16S0B4 | |
ITS0B4AMF02 | |||||
6RD4 | 6LVD6 | 16S0C4 | ITS0C4AMF03 | ||
16S0D4 | ITS0D4 | AMF04 | |||
6LVD76RD5 | 16S0E4 | ITS0E4 | AMF05 | ||
16S0F4 | ITS0F4AMF06 | ||||
6RD6 | 16S0C5 | ITS0C5 | |||
16S0D5 | ITS0D5 | ||||
6RD7 | 16S0E5 | ITS0E5 | |||
16S0F5 | ITS0F5 |
MasterMix (make for 16 plates in 50mL conical tube)
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 1400 | 4200 |
0.45 | 10M dNTPs | 1400 | 630 |
0.3 | Kapa HiFi HotStart DNA Pol | 1400 | 420 |
7.25 | HPLC H2O | 1400 | 10,150 |
11 | Total Volume | 1400 | 15400 |
Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “NS6 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 500 | 1500 |
0.45 | 10M dNTPs | 500 | 225 |
0.3 | Kapa HiFi HotStart DNA Pol | 500 | 150 |
7.25 | HPLC H2O | 500 | 3625 |
11 | Total Volume | 500 | 5500 |
Run on Thermocycler Program GSAF36 or AMF35:
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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