| Info |
|---|
Samples arrived 05-05-2022. Samples checked in against list 7-13-2022. Plates 1-3 have been extracted, normalized, PCRed, quantified and normalized by 7-15-22. 16S for 2TRNL1 was re-PCRed with G4 and H4 because there was low amplification with A5 and B5. |
Extractions
- Transfer fecal material to labeled bead tubes. Weigh first few samples to approximate amount of material ~200mg
- Freeze dry samples
- Extract using MN Nucleospin 96 Stool kits following spin speeds from MN Nucleospin 96 Soil kits
- Transfer DNAs to plate
- Transfer 30 ul as working aliquot
- Add 4 samples of 5 ng/ul Mock Community Only and 4 samples of half mock community half DNA from a single plant sample DNA to column 9(first empty column) of 2TRNL8
- Add ISD and coligos
- Normalize
PCR MasterMix (28 Plates)
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 19002690 | 58,700070 |
0.45 | 10M dNTPs | 19002690 | 8551211 |
0.3 | Kapa HiFi HotStart DNA Pol | 19002690 | 570807 |
7.25 | HPLC H2O | 19002690 | 1319,775502 |
11 | Total Volume | 19002690 | 2029,900590 |
Add 11 ul to each well of a hard shell, full skirt plate. Add 2 uL of primers and 2uL of template to each well.
Plate | TRNL Primers | 16S Primers |
|---|---|---|
2TRNL1 | TRNL01 | 16S0A116S0A5 |
TRNL02 | 16S0B116S0B5 | |
2TRNL2 | TRNL03 | 16S0C116S0C5 |
TRNL04 | 16S0D116S0D5 | |
2TRNL3 | TRNL05 | 16S0E116S0E5 |
TRNL06 | 16S0F116S0F5 | |
2TRNL4 | TRNL07 | 16S0G116S0G5 |
TRNL08 | 16S0H116S0H5 | |
2TRNL5 | TRNL09 | 16S0A216S0A6 |
TRNL10 | 16S0B216S0B6 | |
2TRNL6 | TRNL11 | 16S0C216S0C6 |
TRNL12 | 16S0D216S0D6 | |
2TRNL7 | TRNL13 | 16S0E216S0E6 |
TRNL14 | 16S0F216S0F6 |
Template Format:
Run TRNL plates on thermocycler program TRNL_T*:
...