| Info |
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Reilly Dibner and Cynthia Weinig have requested these 53 samples be re-prepped from NovaSeq1 (A-C). Avoid using MID plates 16S0C8, 16S0D8, ITS0C8, ITS0D8. |
- Pull Plates Dibner_1-95_Norm, Dibner_96-190_Norm, MP101, and MP102 and allow to Thaw
- Tap plates to drop liquid, Spin to drop liquid
- SpeedVac to dry samples
- Resuspend Dibner_1-95_Norm and Dibner_96-190_Norm with 12 ul of ultra pure water
- Resuspend MP101 and MP102 with 25 ul ultra pure water
- Transfer the below samples to a new plate
plate_name | plate_position | sample_name | read_number | row | column |
|---|---|---|---|---|---|
Dibner_1-95 | A1 | cp3_nb2_aug_19_19 | 2263 | A | 1 |
Dibner_1-95 | B1 | cp1_sf4_oct_18_19 | 1807 | B | 1 |
Dibner_1-95 | D1 | cp3_nb2_jun_24_19 | 7372 | D | 1 |
Dibner_1-95 | G1 | cp1_nb2_sep_23_19 | 512 | G | 1 |
Dibner_1-95 | H1 | cp1_nb2_may_22_19 | 1012 | H | 1 |
Dibner_1-95 | A2 | cp1_nb2_jul_23_19 | 12446 | A | 2 |
Dibner_1-95 | B2 | cp1_sf1_jun_20_19 | 1338 | B | 2 |
Dibner_1-95 | C2 | cp1_sf2_sep_23_19 | 1610 | C | 2 |
Dibner_1-95 | D2 | cp1_uf7_jul_23_19 | 10130 | D | 2 |
Dibner_1-95 | E2 | cp3_uf1_aug_19_19 | 2841 | E | 2 |
Dibner_1-95 | F2 | cp1_nb1_aug_19_19 | 2251 | F | 2 |
Dibner_1-95 | G2 | cp1_uf6_may_22_19 | 6034 | G | 2 |
Dibner_1-95 | H2 | cp1_uf3_apr_08_19 | 3132 | H | 2 |
Dibner_1-95 | B3 | cp1_uf6_sep_23_19 | 9789 | B | 3 |
Dibner_1-95 | A4 | cp1_uf4_apr_08_19 | 1701 | A | 4 |
Dibner_1-95 | B4 | cp1_uf5_apr_15_19 | 3837 | B | 4 |
Dibner_1-95 | D4 | 1B_nana_nov_22_19 | 657 | D | 4 |
Dibner_1-95 | G4 | cp1_uf6_aug_19_19 | 742 | G | 4 |
Dibner_1-95 | A5 | cp2_sf3_aug_20_19 | 658 | A | 5 |
Dibner_1-95 | B5 | cp3_nb1_jun_24_19 | 19290 | B | 5 |
Dibner_1-95 | C5 | cp3_uf2_jun_24_19 | 6000 | C | 5 |
Dibner_1-95 | A6 | cp2_sf3_jun_21_19 | 9145 | A | 6 |
Dibner_1-95 | H6 | 6B_nana_nov_22_19 | 141 | H | 6 |
Dibner_1-95 | A7 | cp1_nb4_sep_23_19 | 697 | A | 7 |
Dibner_1-95 | E7 | cp1_nb1_jun_20_19 | 3107 | E | 7 |
Dibner_1-95 | B8 | cp1_sf3_jun_20_19 | 3451 | B | 8 |
Dibner_1-95 |
| cp1_sf4_jun_20_19 | 1626 | B | 8 |
Dibner_1-95 | C8 | cp1_uf5_jul_23_19 | 10685 | C | 8 |
Dibner_1-95 | G8 | cp1_nb2_mar_15_19 | 15710 | G | 8 |
Dibner_1-95 | D9 | cp1_sf3_aug_19_19 | 7424 | D | 9 |
Dibner_1-95 | H9 | 5D_nana_nov_22_19 | 3085 | H | 9 |
Dibner_1-95 | B10 | cp1_nb2_aug_19_19 | 17950 | B | 10 |
Dibner_1-95 | H10 | cp2_nb2_jun_21_19 | 13715 | H | 10 |
Dibner_1-95 | E11 | cp1_sf5_may_22_19 | 2134 | E | 11 |
Dibner_1-95 | C12 | cp1_sf4_jul_23_19 | 1037 | C | 12 |
Dibner_1-95 | H12 | blank | 21 | H | 12 |
Dibner_96-190 | E3 | cp1_nb5_apr_15_19 | 14423 | E | 3 |
Dibner_96-190 | C4 | cp1_uf5_jun_20_19 | 5544 | C | 4 |
Dibner_96-190 | B5 | cp1_sf1_sep_23_19 | 1435 | B | 5 |
MP101 | G1 | MP101_G1 | 1793 | G | 1 |
MP101 | H1 | MP101_H1 | 50 | H | 1 |
MP101 | H4 | MP101_H4 | 212 | H | 4 |
MP101 | A11 | MP101_A11 | 10565 | A | 11 |
MP102 | A1 | MP102_A1 | 1525 | A | 1 |
MP102 | E1 | MP102_E1 | 6866 | E | 1 |
MP102 | B2 | MP102_B2 | 491 | B | 2 |
MP102 | H2 | MP102_H2 | 1195 | H | 2 |
MP102 | B3 | MP102_B3 | 740 | B | 3 |
MP102 | H3 | MP102_H3 | 5542 | H | 3 |
MP102 | D5 | MP102_D5 | 758 | D | 5 |
MP102 | D8 | MP102_D8 | 374 | D | 8 |
MP102 | H9 | MP102_H9 | 453 | H | 9 |
MP102 | G12 | MP102_G12 | 8669 | G | 12 |
Well Position | Sample |
|---|---|
A01 | cp3_nb2_aug_19_19 |
B01 | cp1_sf4_oct_18_19 |
C01 | cp3_nb2_jun_24_19 |
D01 | cp1_nb2_sep_23_19 |
E01 | cp1_nb2_may_22_19 |
F01 | cp1_nb2_jul_23_19 |
G01 | cp1_sf1_jun_20_19 |
H01 | cp1_sf2_sep_23_19 |
A02 | cp1_uf7_jul_23_19 |
B02 | cp3_uf1_aug_19_19 |
C02 | cp1_nb1_aug_19_19 |
D02 | cp1_uf6_may_22_19 |
E02 | cp1_uf3_apr_08_19 |
F02 | cp1_uf6_sep_23_19 |
G02 | cp1_uf4_apr_08_19 |
H02 | cp1_uf5_apr_15_19 |
A03 | 1B_nana_nov_22_19 |
B03 | cp1_uf6_aug_19_19 |
C03 | cp2_sf3_aug_20_19 |
D03 | cp3_nb1_jun_24_19 |
E03 | cp3_uf2_jun_24_19 |
F03 | cp2_sf3_jun_21_19 |
G03 | 6B_nana_nov_22_19 |
H03 | cp1_nb4_sep_23_19 |
A04 | cp1_nb1_jun_20_19 |
B04 | cp1_sf3_jun_20_19 |
C04 | cp1_sf4_jun_20_19 |
D04 | cp1_uf5_jul_23_19 |
E04 | cp1_nb2_mar_15_19 |
F04 | cp1_sf3_aug_19_19 |
G04 | 5D_nana_nov_22_19 |
H04 | cp1_nb2_aug_19_19 |
A05 | cp2_nb2_jun_21_19 |
B05 | cp1_sf5_may_22_19 |
C05 | cp1_sf4_jul_23_19 |
D05 | blank |
E05 | cp1_nb5_apr_15_19 |
F05 | cp1_uf5_jun_20_19 |
G05 | cp1_sf1_sep_23_19 |
H05 | MP101_G1 |
A06 | MP101_H1 |
B06 | MP101_H4 |
C06 | MP101_A11 |
D06 | MP102_A1 |
E06 | MP102_E1 |
F06 | MP102_B2 |
G06 | MP102_H2 |
H06 | MP102_B3 |
A07 | MP102_H3 |
B07 | MP102_D5 |
C07 | MP102_D8 |
D07 | MP102_H9 |
E07 | MP102_G12 |
F07 | Mock Community |
G07 | Mock Community |
H07 | Mock Community |
PCR1 (Manual):
Create MasterMix:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 224 | 840 |
0.45 | 10M dNTPs | 224 | 126 |
0.3 | Kapa HiFi HotStart DNA Pol | 224 | 84 |
3.25 | HPLC H2O | 224 | 910 |
7 | Total Volume | 224 | 1960 |
...
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00 |
98 | 15X | 0:30 |
62 | 15X | 0:30 |
72 | 15X | 0:30 |
72 | 1X | 5:00 |
4 | 1X | 0:00 |
PCR2 Mastermix Preparation:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HF Buffer | 112 | 540 |
0.45 | 10M dNTPs | 112 | 81 |
0.3 | Kapa HiFi HotStart DNA Pol | 112 | 54 |
0.5 ul | 5 uM F and R FlowCell Primers | 112 | 90 |
0.75 | HPLC H2O | 112 | 135 |
5 | Total Volume | 112 | 900 |
Add 5 ul master mix to all wells of 4 hard shell full skirted plates. Seal with tape seals and store in refrigerator.
Intermediate Cleanup:
The majority of this was done on the Nimbus platform using “AxyPrep MagBead PCR1 No MM”
...
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 (Plate1 PCR1 MIDPlate1 MIDPlate2)
Transfer 10 ul from transparent plate to “PCR2” plate with Mastermix already added.
Label Plate1 PCR2 MIDPlate1 MIDPlate2
Seal “PCR2”s with bubble strips and run on thermocycler 35GSAF2 program
...
Temp C
...
Cycles
...
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 |
...
*PCR2 actually had 20 cycles, not 19
Final Cleanup:
Equilibrate Beads to room Temperature
...