PCR
MasterMix for the new primer plates was made according to https://microcollaborative.atlassian.net/wiki/spaces/MICLAB/pages/2002321455 on 12-09-2022.
MasterMix for the previously unused original primer plates was made according to https://microcollaborative.atlassian.net/wiki/spaces/MICLAB/pages/1979383819 on 10-20-22.
9 ul of the top MM was aliquoted into each well for plates run with the new primers.
11 ul of the second MM was aliquoted into each well for plates run with unused original primers (5AL1_Norm and 5AL2_Norm’s per plates).
The Nimbus was used to array the new fwd primers directly into the MM via the program PcrPrep_LongPrimer_Tracking_FullyAdjustable
rev primers were stamped using the 96 channel pipette
2 ul of the unused primer plates was added to the appropriate wells (those with 11 ul of the 2nd MM)
Resulting MM plates were sealed and labeled with the actual or equivalent MID plate and F# and R# to indicate what was added and from where
2 ul of template was added to each of these MM plates matching the pattern below:
Template Plate | Unused Original Primer Plate | Fwd Primer Plate | Rev Primer Plate Used | Equivalent MID map |
|---|---|---|---|---|
5AL1_Norm | 16S0A12 16S0B12 | NA | NA | 16S0A12 16S0B12 |
ITS0A12 ITS0B12 | NA | NA | ITS0A12 ITS0B12 | |
5AL2_Norm | 16S0C12 16S0D12 | NA | NA | 16S0C12 16S0D12 |
ITS0C12 ITS0D12 | NA | NA | ITS0C12 ITS0D12 | |
5AL3_Norm | NA | long16Sfwd120922_Working5 | long16Sfwd120922_Working1 | 16S0A1 16S0B1 |
NA | longITSfwd120922_Working5 | longITSfwd120922_Working1 | ITS0A1 ITS0B1 | |
5AL4_Norm | NA | long16Sfwd120922_Working5 | long16Sfwd120922_Working1 | 16S0C1 16S0D1 |
NA | longITSfwd120922_Working5 | longITSfwd120922_Working1 | ITS0C1 ITS0D1 | |
5AL5_Norm | NA | long16Sfwd120922_Working5 | long16Sfwd120922_Working1 | 16S0E1 16S0F1 |
NA | longITSfwd120922_Working5 | longITSfwd120922_Working1 | ITS0E1 ITS0F1 | |
5AL6_Norm | NA | long16Sfwd120922_Working5 | long16Sfwd120922_Working1 | 16S0G1 16S0H1 |
NA | longITSfwd120922_Working5 | longITSfwd120922_Working1 | ITS0G1 ITS0H1 | |
5AL7_Norm | NA | long16Sfwd120922_Working5 | long16Sfwd120922_Working1 | 16S0A2 16S0B2 |
NA | longITSfwd120922_Working5 | longITSfwd120922_Working1 | ITS0A2 ITS0B2 | |
5AL8_Norm | NA | long16Sfwd120922_Working5 | long16Sfwd120922_Working1 | 16S0C2 16S0D2 |
NA | longITSfwd120922_Working5 | longITSfwd120922_Working1 | ITS0C2 ITS0D2 |
Plates were then bubble sealed and run 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:
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
Post PCR Normalization
Cleaned plates were quantified via absorbance
Data were fed to Nimbus program Normalization_Tracking_Adaptive and adjusted to 5 ng/ul or less