4 full plates plus 7 columns on a 5th. We will add 8 lambda samples as positive controls and to make the partial plate easier to work with the automation. Hold off on sequencing unless instructed to proceed!
Used SpeedVac to evaporate all liquid from all samples. Resuspended in 30 ul UPW.
Restriction Digestion
(Keep MM and reaction plates on ice)
...
Reagent | ul/rxn | rxns | ul needed (1.6x) | |
|---|---|---|---|---|
MseI oligo | 1 | 700 | 700 | 100 |
H2O | 0.112 | 700 | 78.4 | 11.2 |
10x T4 Buffer | 0.1 | 700 | 70 | 10 |
5M NaCl | 0.01 | 700 | 7 | 1 |
1 mg/ml BSA | 0.05 | 700 | 35 | 5 |
T4 DNA ligase | 0.1675 | 700 | 117.25 | 17 |
Total | 1.4 | 700 | 980 | 140 |
Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate. Leave out at room temperature for ~2 hours.
...
Reagent | ul/rxn | rxns | ul needed (x1.4) | |
|---|---|---|---|---|
H2O | 9.52 | 350 | 3332 | 952 |
5x iProof buffer | 4 | 350 | 1400 | 400 |
10 mM dNTPs | 0.4 | 350 | 140 | 40 |
50 mM MgCl2 | 0.4 | 350 | 140 | 40 |
5 uM Illumina Primers | 1.33 | 350 | 466 | 465.5133 |
iProof TAQ | 0.2 | 350 | 70 | 20 |
DMSO | 0.15 | 350 | 52.5 | 15 |
total | 16 | 350 | 5600 | 288001600 |
Add 16 ul of PCR1 MM to each well of 2+12(for 4Trout) new hard shell PCR plates with AssistPlus program 16ulMMDisp on 125ul ViaFlo
...
Reagent | ul/rxn | rxns | ul needed (x 1.6) | |
5x Iproof buffer | 0.425 | 350 | 149 | 42.5 |
10 mM dNTPs | 0.4 | 350 | 140 | 40 |
Primers | 1.33 | 350 | 466 | 133 |
Total | 2.155 | 350 | 755 |
Then continue with the last four unlisted steps for GBS1 from above:
...
Template | EcoR1 MID plate | Pool Plate | Library | Norm to | Sequencer |
|---|---|---|---|---|---|
PSMA1 | 2 | ||||
PSMA2 | 3 | ||||
PSMA3 | 4 | ||||
PSMA4* | 1 | ||||
ANBO1 | 5 |
Size Select at 275-375 bp on Pippin Prep 2% no dye gel
Before Size Selection:
...
250-500 bp selection:
...
275-375 bp selection:
...