Reagents
Materials & Locations
| Reagent | Location |
|---|
Step 1
Oligo Phosphorylation → Annealing
Master Mix
| Reagent | Per rxn (µL) | Master mix (µL) |
|---|
Aliquot 4.0 µL/tube from master mix, then add 0.5 µL UP + 0.5 µL DW oligo individually.
Cycling Program
Phosphorylation/Annealing
Stage 1
Phosphorylation
°C ʹ
Stage 2
Denaturation
°C ʹ
Stage 3
Renaturation (slow cooling)
target °C
Ramp rate
°C/s
Hold
Indefinite hold
°C
Step 2
Assembly (Golden Gate Ligation)
First, dilute step 1 product 1:40: add 195 µL mqH2O to each step 1 tube (5 µL → 200 µL total). Use 0.5 µL of this dilution per assembly reaction.
Master Mix
| Reagent | Per rxn (µL) | Master mix (µL) |
|---|
Aliquot 4.5 µL/tube from master mix, then add 0.5 µL of 1/40 diluted oligo pair per tube.
Cycling Program
"goldgate" — DE folder
Cycles
× cycles
Step 1 – Digestion
°C ʹ
Step 2 – Ligation
°C ʹ
Stage 2
Inactivation
°C ʹ
Hold
Indefinite hold
°C
Step 3
Transformation (NEBStable Competent Cells)
Notes
Experiment Notes
saved