25vanD1
- Gregg Randolph
Last wells of the last column of each plate are Mock Community, TE with ISD, and just TE.
Check reagents for contam:
PCR contam:
qPCRed and taped TE, 10 pg/ul ISD, and 0.001 pg/ul ISD with Illumina primers
All sample curves below NTCs
DNA contam:
Ran 2 samples each of TE, H2O, loading ISD, and Mock Community using below MM and PCR parameters with A1-A1 16S primers.
No evidence shown for 16S DNA or PCR contamination
MasterMix
ul/rxn | Reagent | # of rxns | ul needed |
|
|---|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 2000 | 6000 | 975 |
0.45 | 10M dNTPs | 2000 | 900 | 146 |
0.3 | Kapa HiFi HotStart DNA Pol | 2000 | 600 | 98 |
5.25 | HPLC H2O | 2000 | 10500 | 1706 |
9 | Total Volume | 2000 | 18000 |
|
Add 9 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “NS5 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of each of the primers).
Plate | AMF Primer | ITS Primer | 16S Primer |
|---|---|---|---|
25LVD1_1 | AMF02 | ITS0D12 | 16S0C4 |
ITS0B10 | 16S0B10 | ||
25LVD1_2 | AMF01 | ITS0H11 | 16S0F11 |
ITS0A12 | 16S0H12 | ||
25LVD1_3 | AMF03 | ITS0F11 | 16S0A11 |
25LVD1_4 | AMF04 | ITS0E10 |
|
ITS0H10 |
| ||
25LVD1_5 | AMF05 | ITS0B11 |
|
qPCR and Sequencing
16S has 216, ITS has 360, AMF has 360
16S
Pool concentration = 9.17
1nM Dilution = 23.6 ul with 192.4
Load =
ITS
Pool concentration = 8.42
1nM Dilution = 42.8 with 317.2
Load =
AMF
Pool concentration = 8.29
1nM Dilution = 43.4 with 316.6
Load =