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ITS Coligo: 5'-CTTGGTCATTTAGAGGAAGTAAT AACAACAACAACC CGTAGCTACTTCTTGCGTCG-3'
Cross contamination oligo creation Coligo creation and quality control
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Synthgene Structure: 5'-LocusPrimer Nonsense InverseLocusPrimer-3'
ITS Synthgene:
CTTGGTCATTTAGAGGAAGTAATGCCACAGATACGTACCGCTCATAACGCGAACCGAAGCGCAGTAGAAGTACTCCGTATCCTACCTCGGTCGTGGTTTAGGCTATCGACATCTTGCATGGGCTTCCCTAGTGAACTCTTGGGATGTGCATCGATGAAGAACGCAGC
Alignment of primers and synthetic amplicon control molecules (synthgenes): Synthgene_detective.pdf (also on OneDrive).
16S Synthgene:
GTGCCAGCAGCCGCGGTAAGCCACAGATACGTACCGCTCATAACGCGAACCGAAGCGCAGTAGAAGTACTCCGTATCCTACCTCGGTCGTGGTTTAGGCTATCGACATCTTGCATGGGCTTCCCTAGTGAACTCTTGGGATGTATTAGATACCCTAGTAGTCC
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Add 6 ul appropriate 0.25 uM paired primers Molecular IDentifier (MID) Plate:______________
16S Primer Base
515F (5’-GTGYCAGCMGCCGC GGTAA-3’) (Parada et al. 2016)
806R (5’-GGACTACNVGGGTWTCTAAT-3’) (Apprill et al. 2015)
ITS Primer Base
ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3')(Gardes et al 1993)
ITS2 (3'-CGTAGCTACTTCTTGCGTCG-5') (White et al 1990)
Primer structure
5'-
PARTIAL_ILLUMINA_FLOWCELL_SEQbarcodeGTGYCAGCMGCCGCGGTAA-3'5'-
PARTIAL_ILLUMINA_FLOWCELL_SEQbarcodeGGACTACHVGGGTWTCTAAT-3'5'-
PARTIAL_ILLUMINA_FLOWCELL_SEQbarcodeCTTGGTCATTTAGAGGAAGTAA-3'5'-
PARTIAL_ILLUMINA_FLOWCELL_SEQbarcodeGCTGCGTTCTTCATCGATGC-3'
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Remove sample plate from magnet plate.
Add 30 40 ul H2OTE; pipette mix 10+ times. Incubate 2 minutes at RT.
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ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 60 | 210 |
0.45 | 10M dNTPs | 60 | 31.5 |
0.3 | Kapa HiFi HotStart DNA Pol | 60 | 21 |
0.5 ul | 10 uM F and R FlowCell Primers | 60 | 35 |
0.75 | HPLC H2O | 60 | 52.5 |
5 | Total Volume | 60 | 350 |
FlowCell Primers
AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTC
CAAGCAGAAGACGGCATACGAGATGTCTCGTGGGCTCGG
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Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00* |
98 | 19X | 0:30 |
55* | 19X | 0:30 |
72 | 19X | 0:30 |
72 | 1X | 5:00 |
4 | 1X | 0:00 |
Purify samples using GSAF’s second modified MagBead protocol:
Equilibrate Beads to room Temperature
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Remove sample plate from magnet plate.
Add 30 40 ul TE; pipette mix 10 times
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Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Transfer 30 40 ul to a clean PCR plate.
Use Synergy HTX’s Take 3 Trio to quantify DNA via absorbance.
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Normalize PCR’s via absorbance concentrations. Gregg Randolph What concentration did you normalize to? Or has this not been finalized yet?
Pool normalized PCRs
Check Pool’s molar concentration via qPCR:
Dilute Pool 1:1000
Include NTC and Standards (20 pm, 2 pm, 0.2, 0.02pm, 0.002pm, 0.0002pm)
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