1EMF
~90 samples from Kelly Goodwin looking at AMF and EMF using AMF and ITS primers
Added MC in G12 and filled the rest with NTC for a full plate
MasterMix (make for 16 plates in 50mL conical tube)
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 420 | 1260 |
0.45 | 10M dNTPs | 420 | 189 |
0.3 | Kapa HiFi HotStart DNA Pol | 420 | 126 |
7.25 | HPLC H2O | 420 | 3045 |
11 | Total Volume | 420 | 4620 |
Add 9 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plate | ITS Primer | AMF Primer |
|---|---|---|
1EMF1 | ITS0H10 | AMF05 |
ITS0H11 | AMF06 |
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:
Plates were cleaned up utilizing the BenchSmart 96 channel pipette:
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 4 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)
Normalize to 5 ng/ul
Store in -20 until ready to qPCR
Tapestation
2 samples, 1 Mock Community positive control, and one NTC were checked for amplification for both loci. They matched expectations.
Mock Community AMF:
NTC AMF:
Sample from G11 AMF:
Sample from H11 AMF:
Mock Community ITS:
NTC ITS:
Sample from G11 ITS:
Sample from H11 ITS:
qPCR and dilution
Will be combined with 1EAST and SeqWellLambda2.
SeqWellLambda2 has a concentration of 7 nM
1EMF1_AMF has a concentration of 3.92 nM
1EMF_ITS has a concentration of 1.17 nM
1EAST has a concentration of 0.5 nM
2.2 ul SeqWellLambda2 will give us 300 pM in 50 ul
20 ul 1EAST will give us 200 pM in 50 ul
3.2 ul 1EMF_AMF will give us 250 pM in 50 ul
7.2 ul 1EMF_ITS will give us 250 pM in 50 ul
Add 17.4ul RSB to bring us to 1nM total