16S V4 Project with PNA blockers
Dilute Stock PNAs to working concentration
50 uM PNA blockers mPNA and pPNA
12 uM allows for 2.5 ul per PNA to reach 2 uM and 0.625 ul for 0.5 uM
24 ul PNA in 76 ul TE makes 100 ul 12uM PNAs
MasterMix for standard
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 150 | 450 |
0.45 | 10M dNTPs | 150 | 68 |
0.3 | Kapa HiFi HotStart DNA Pol | 150 | 45 |
5.25 | HPLC H2O | 150 | 787 |
9 | Total Volume | 150 | 1350 |
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).
MasterMix for PNA work
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 50 | 150 |
0.45 | 10M dNTPs | 50 | 22.5 |
0.3 | Kapa HiFi HotStart DNA Pol | 50 | 15 |
0.25 | HPLC H2O | 50 | 12.5 |
4 | Total Volume | 50 | 200 |
Add 4 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).