...
Size Selection: 300-500
Quantify and normalize samples:
...
Add 3 ul Digestion MM to 3 plates using the 8 channel
Reagent | ul/rxn | rxns | ul needed (1.5x) |
10x T4 Buffer | 1.15 |
552 |
920 | |
5M NaCl | 0.12 |
552 |
96 | |
1 mg/ml BSA | 0.6 |
552 |
480 | |
H2O | 0.73 |
552 |
584 | |
MseI (enzyme) | 0.12 |
552 |
96 | |
EcoR1 (enzyme) | 0.28 |
552 |
224 | ||
Total | 3 | 552 |
2400 |
Add 6 ul template to each plate with Benchsmart. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 65C in EcoBGC oven for 1 hour.
...
Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.
Reagent | ul/rxn | rxns | ul needed (1.6x) |
|---|---|---|---|
MseI oligo | 1 |
552 |
800 | |
H2O | 0.112 |
552 |
89.6 | |
10x T4 Buffer | 0.1 |
552 |
80 | |
5M NaCl | 0.01 |
552 |
8 | |
1 mg/ml BSA | 0.05 |
552 |
40 | |
T4 DNA ligase | 0.1675 |
552 |
134 | ||
Total | 1.4 | 552 |
1120 |
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 |
Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel
...
Seal, Vortex, Spin down, and then run on Thermocycler program: TANK1 TANKGBS:
SPECIAL CYCLING PROGRAM!!!! ONLY 20 CYCLES!!!!
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
...
Reagent
...
ul/rxn
...
rxns
...
ul needed (x 1.6)
...
5x Iproof buffer
...
0.425
...
272
...
136
...
10 mM dNTPs
...
0.4
...
272
...
128
...
Primers
...
1.33
...
272
...
426
...
Total
...
2.155
...
272
...
690
Then continue with the last four unlisted steps for GBS1 from above:
Temp C
Cycles
Time
98 | 1X | 3:00 |
60 | 1X | 2:00 |
72 | 1X | 10:00 |
4 | 1X | 0:00 |
*pause step