| Info |
|---|
Received New DNAs 9-6-2022 Digests started 9-12-2022 Ligation started 9-14-2022 PCR started 9-15-2022 |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | L3_PJD83 | L6_PJD86 | L1_PJD94 | L2_PJD91 | ||||||||
B | H6_PJD98 | H2_PJD95 | L7_PJD97 | I10_PJD75 | ||||||||
C | B2377_PJD57 | H5_PJD82 | I7_PJD80 | H4_PJD89 | ||||||||
D | L9_PJD99 | I1_PJD73 | L4_PJD87 | I14_PJD81 | ||||||||
E | B2461_PJD60 | L5_PJD77 | I9_PJD74 | I4_PJD85 | ||||||||
F | B2437_PJD58 | I13_PJD92 | I11_PJD90 | I5_PJD79 | ||||||||
G | I12_PJD96 | I6_PJD88 | I2_PJD84 | H1_PJD76 | ||||||||
H | B2453_PJD59 | I15_PJD78 | L10_PJD61 | H3_PJD93 |
...
Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.
Template | EcoR1 MID plate |
|---|---|
JJ_AW_1 (1SMIN11BUN2 (1PJD2) | EcoR1 MID plate 1 |
JJ_AW_2 (1SMIN2) | EcoR1 MID plate 2 |
PJDB_JJF (1BUN1) | EcoR1 MID plate 3 |
4 |
Label reaction plates with MID plate used.
...
Create working pools. Load Pool A1 into columns 1-6 and A2 into columns 7-12 of the pooling plate.
Pool | Plate1 Col 1-6 | Plate1 Col 7-122 | Plate2 Plate1 Col 1-63 | Plate2 Col 7-12 | 1Smin Pool A14 |
|---|---|---|---|---|---|
Pool | Plate1 Col 1-6 | Plate1 Col 12-7 | Plate2 Col 1-6 | Plate2 Col 12-7 | 1Smin Pool A2 |
|---|
1
2
3
4
5
6
7
8
9
10
11
12
A
PLT1_Col1
PLT1_Col7
PLT2_Col1
PLT2_Col7
PLT1_Col2
PLT1_Col8
PLT2_Col2
PLT2_Col8
PLT1_Col3
PLT1_Col9
PLT2_Col3
PLT2_Col9
PLT1_Col4
PLT1_Col10
PLT2_Col4
PLT2_Col10
PLT1_Col5
PLT1_Col11
PLT2_Col5
PLT2_Col11
PLT1_Col6
PLT1_Col12
PLT2_Col6
PLT2_Col12
PLT1_Col1
PLT1_Col12
PLT2_Col1
PLT2_Col12
PLT1_Col2
PLT1_Col11
PLT2_Col2
PLT2_Col11
PLT1_Col3
PLT1_Col10
PLT2_Col3
PLT2_Col10
PLT1_Col4
PLT1_Col9
PLT2_Col4
PLT2_Col9
PLT1_Col5
PLT1_Col8
PLT2_Col5
PLT2_Col8
PLT1_Col6
PLT1_Col7
PLT2_Col6
PLT2_Col7
B
C
D
E
F
G
H
Pool
Plate3 Rows A-B
Plate3 Rows C-D
Plate3 Rows E-F
Plate3 Rows G-H
PJDA
Pool
Plate3 Rows A-B
Plate3 Rows D-C
Plate3 Rows E-F
Plate3 Rows H-G
PJDB
Pool
Plate3
Plate3
Plate3
Plate3
Mix1
A7, C7, E7, G7
B7, D7, F7, H7
Pool
Plate3
Plate3
Plate3
Plate3
Mix2
A7-D7
E7-H7
1
2
3
4
5
6
7
8
9
10
11
12
A
PLT3_Row A, Row C, Row E, Row G
PLT3_Row B, Row D, Row F, Row H
PLT3_Row A, Row D, Row E, Row H
PLT3_Row B, Row C, Row F, Row G
PLT3_A7, C7, E7, G7
B
PLT3_B7, D7, F7, H7
C
PLT3_A7, B7, C7, D7
D
PLT3_E7, F7, G7, H7
E
F
G
H
2 (flipped) | Plate1 Col 3 | Plate2 Col 4(flipped) | ||
|---|---|---|---|---|
PCR1:
Make MM1 in a 1.5mL tube:
Reagent | ul/rxn | rxns (x1.3) | ul needed |
|---|---|---|---|
H2O | 9.52 | 14816 | 1837.4238 |
5x iProof buffer | 4 | 14816 | 772100 |
10 mM dNTPs | 0.4 | 14816 | 77.210 |
50 mM MgCl2 | 0.4 | 14816 | 77.210 |
5 uM Illumina Primers | 1.33 | 14816 | 25633.725 |
iProof TAQ | 0.2 | 14816 | 38.65 |
DMSO | 0.15 | 14816 | 293.75 |
total | 16 | 14816 | 3088400 |
Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel
...
Reagent | ul/rxn | rxns (x1.6) | ul needed |
5x Iproof buffer | 0.425 | 14816 | 10010.72 |
10 mM dNTPs | 0.4 | 14816 | 949.86 |
Primers | 1.33 | 14816 | 31531.29 |
Total | 2.155 | 14816 | 51051.7 |
Then continue with the last four unlisted steps for GBS1 from above:
...