BrockPna
- Gregg Randolph
16S V4 Project with PNA blockers
Dilute Stock PNAs to working concentration
50 uM PNA blockers mPNA and pPNA
6 uM allows for 5 ul per combined PNA to reach 2 uM and 1.25 ul for 0.5 uM
12 ul each PNA in 76 ul TE makes 100 ul 6uM PNAs with both m and p
Dilute DNAs to 10 ng/ul
Use concentrations provided
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 each 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 |
2.25 | HPLC H2O | 50 | 112.5 |
6 | Total Volume | 50 | 300 |
Add 6 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “NS5 PCR”. ( one needs 2 ul of template and 2 ul of the primers).
PNA volumes
|
|
|
|
|---|---|---|---|
1.25 | 0 | 1.25 | 0 |
1.25 | 2.5 | 1.25 | 2.5 |
1.25 | 5 | 1.25 | 5 |
1.25 | 0 | 1.25 | 0 |
1.25 | 2.5 | 1.25 | 2.5 |
1.25 | 5 | 1.25 | 5 |
1.25 |
| 1.25 |
|
1.25 |
| 1.25 |
|
Water volumes
1 | 2 | 3 | 4 |
|---|---|---|---|
3.75 | 5 | 3.75 | 5 |
3.75 | 2.5 | 3.75 | 2.5 |
3.75 | 0 | 3.75 | 0 |
3.75 | 5 | 3.75 | 5 |
3.75 | 2.5 | 3.75 | 2.5 |
3.75 | 0 | 3.75 | 0 |
3.75 |
| 3.75 |
|
3.75 |
| 3.75 |
|
PCR
Run STD 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 |
Run PNA on PNA_GSAF
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00* |
98 | 36X | 0:30 |
78 | 36X | 0:15 |
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”
Manually, it was done:
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)
Store in -20 until ready to qPCR
TapeStation
Pool:
Samples:
Pool, followed by Brock 2, column 2 (sample 5 is the blank)
Blank:
qPCR
45 nM
2.2 ul sample plus 97.8 ul RSB to make 1 nM
Sequence Dilution
Dilute to 1 nM:
2.2 ul sample plus 97.8 ul RSB
Add 10% PhiX and dilute to 65 pM:
13 ul sample plus 1.3 ul PhiX plus 185.7 RSB