...
Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.
Add 40 ul TE to all wells
Pool Within Plates:
Use Assist Plus to pool within plate.
Plate 6 with Plate1 : PCR1+Pool1
Use 8Conly to pool 8 columns of Plate6 into 4 columns
Pool 9th column by twos into G5 and H5
Pool Plate 1 by hand
Pool first two columns in sets of 4 wells into A5-D5
Pool third column by twos into E5 and F5
Plate 2 with Plate 3: PCR2+Pool2
Plate 4 with Plate 5: PCR3+Pool3
Plate 7 with Plate8: PCR4+Pool4
Move bottom half of Plate 8 to top of columns 5-8
Move top half of Plate 7 to bottom of columns 6-10
Use 8Conly on both
Pool columns 9 and 10 of Plate7 by twos into column 5
PCR1:
Reagent | ul/rxn | rxns | ul needed (x1.4) |
|---|---|---|---|
H2O | 9.52 | 276 | 2856 |
5x iProof buffer | 4 | 276 | 1200 |
10 mM dNTPs | 0.4 | 276 | 120 |
50 mM MgCl2 | 0.4 | 276 | 120 |
5 uM Illumina Primers | 1.33 | 276 | 399 |
iProof TAQ | 0.2 | 276 | 60 |
DMSO | 0.15 | 276 | 45 |
total | 16 | 276 | 4800 |
...
Seal, Vortex, Spin down, and then run on Thermocycler program: TANK1 TANKGBS:
SPECIAL CYCLING PROGRAM!!!! ONLY 20 CYCLES!!!!
Reagent
ul/rxn
rxns
ul needed (x 1.6)
5x Iproof buffer
0.425
276
136
10 mM dNTPs
0.4
276
128
Primers
1.33
276
426
Total
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00 |
98 | 20X | 0:30 |
60 | 20X | 0:30 |
72 | 20X | 0:40 |
72 | 1X | 10:00 |
4* | 1X* | 0:00* |
*pause step
Extra PCR
Add 2.155 ul of the MM directly to the previous PCR
Temp C
Cycles
Time
276
690
Then continue with the last four unlisted steps for GBS1 from above:
98 | 1X | 3:00 |
60 | 1X | 2:00 |
72 | 1X | 10:00 |
4 | 1X | 0:00 |
*pause step