PCR & qPCR Set-Up/Results - Alfalfa 16S/ITS
Prepare two ITS and two 16S PCR plates for the following template plates:
ALF_PLT1_2020_END | ALF_PLT2_2020_END | ALF_PLT3_2020_END | ALF_PLT4_2020_END |
ALF_PLT5_2020_END | ALF_PLT6_2020_END | ALF_PLT7_2020_END | ALF_PLT8_2020_END |
ALF_PLT1_2020_EPI | ALF_PLT2_2020_EPI | ALF_PLT3_2020_EPI | ALF_PLT4_2020_EPI |
ALF_PLT5_2020_EPI | ALF_PLT6_2020_EPI | ALF_PLT7_2020_EPI | ALF_PLT8_2020_EPI |
ALF_PLT9_2020_EPI | ALF_PLT1_2020_SOIL |
|
|
ALF_PLT1_2017_GBS | ALF_PLT2_2017_GBS | ALF_PLT3_2017_GBS | ALF_PLT4_2017_GBS |
ALF_PLT5_2017_GBS | ALF_PLT6_2017_GBS | ALF_PLT7_2017_GBS | ALF_PLT8_2017_GBS |
ALF_PLT9_2017_GBS | ALF_PLT10_2017_GBS | ALF_PLT11_2017_GBS |
|
MasterMix (make for 16 plates in 50mL conical tube)
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 1700 | 5100 |
0.45 | 10M dNTPs | 1700 | 765 |
0.3 | Kapa HiFi HotStart DNA Pol | 1700 | 510 |
7.25 | Ultra Pure H2O | 1700 | 12,325 |
11 | Total Volume | 1700 | 18,700 |
Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “ALF PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plates | 16S MIDs | ITS MIDs |
|---|---|---|
ALF_PLT1_2020_END | long16S0A1 | longITS0A1 |
long16S0B1 | longITS0B1 | |
ALF_PLT2_2020_END | long16S0C1 | longITS0C1 |
long16S0D1 | longITS0D1 | |
ALF_PLT3_2020_END | long16S0E1 | longITS0E1 |
long16S0F1 | longITS0F1 | |
ALF_PLT4_2020_END | long16S0G1 | longITS0G1 |
long16S0H1 | longITS0H1 |
Run 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 |
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)
MasterMix (make for 16 plates in 50mL conical tube)
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 1700 | 5100 |
0.45 | 10M dNTPs | 1700 | 765 |
0.3 | Kapa HiFi HotStart DNA Pol | 1700 | 510 |
7.25 | Ultra Pure H2O | 1700 | 12,325 |
11 | Total Volume | 1700 | 18,700 |
Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “ALF PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plates | 16S MIDs | ITS MIDs |
|---|---|---|
ALF_PLT5_2020_END | long16S0A2 | longITS0A2 |
long16S0B2 | longITS0B2 | |
ALF_PLT6_2020_END | long16S0C2 | longITS0C2 |
long16S0D2 | longITS0D2 | |
ALF_PLT7_2020_END | long16S0E2 | longITS0E2 |
long16S0F2 | longITS0F2 | |
ALF_PLT8_2020_END | long16S0G2 | longITS0G2 |
long16S0H2 | longITS0H2 |
Run 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 |
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)
qPCR ALF_END
Make 1:1000 dilutions from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | ALF_END_PLT1_16S_3B | ALF_END_PLT3_16S_3B | ALF_END_PLT5_16S_3B | ALF_END_PLT7_16S_3B |
|
|
|
|
|
|
| NTC |
B | ALF_END_PLT1_16S_9H | ALF_END_PLT3_16S_9H | ALF_END_PLT5_16S_9H | ALF_END_PLT7_16S_9H |
|
|
|
|
|
| 04.0002 pM Std | 0.0002 pM Std |
C | ALF_END_PLT1_ITS_3B | ALF_END_PLT5_ITS_3B | ALF_END_PLT3_ITS_3B | ALF_END_PLT7_ITS_3B |
|
|
|
|
|
| 0.002 pM Std | 0.002 pM Std |
D | ALF_END_PLT1_ITS_9H | ALF_END_PLT5_ITS_9H | ALF_END_PLT3_ITS_9H | ALF_END_PLT7_ITS_9H |
|
|
|
|
|
| 0.02 pM Std | 0.02 pM Std |
E | ALF_END_PLT2_16S_3B | ALF_END_PLT4_16S_3B | ALF_END_PLT6_16S_3B | ALF_END_PLT8_16S_3B |
|
|
|
|
|
| 0.2 pM Std | 0.2 pM Std |
F | ALF_END_PLT2_16S_9H | ALF_END_PLT4_16S_9H | ALF_END_PLT6_16S_9H | ALF_END_PLT8_16S_9H |
|
|
|
|
|
| 2 pM Std | 2 pM Std |
G | ALF_END_PLT2_ITS_3B | ALF_END_PLT4_ITS_3B | ALF_END_PLT6_ITS_3B | ALF_END_PLT8_ITS_3B |
|
|
|
|
|
| 20 pM Std | 20 pM Std |
H | ALF_END_PLT2_ITS_9H | ALF_END_PLT4_ITS_9H | ALF_END_PLT6_ITS_9H | ALF_END_PLT8_ITS_9H |
|
|
|
|