Use NEBioCalculator 1.17.3 to calculate how much volume of each fragment is needed such that 0.05 pmol of each fragment is added. Use the Oligonucleotide/Gene Database to retrieve fragment lengths. It is recommended to use a table like so in Excel to calculate:
Name
Label
Length (bp)
Mass (ng) for 0.05 pmol
Concentration (ng/ul)
Volume (uL)
Recall C=Vm so V=Cm
Synthesized gene fragments are resuspended to a standard concentration of 10 mM unless otherwise stated.
On ice or a pre-chilled PCR tube rack, add the components into a PCR tube in the following amounts for each reaction:
Add water, then the DNA fragments. Since volumes may be very small, make sure the tip touches the water when pipetting down.
Then add the remaining components, and pipette up and down 3-5 times gently to mix.
For 2-6 fragments (including backbone):
| Component | Volume (µL) |
--- | --- | | NEBridge Ligase Master Mix 3X | 5 | DNA fragments | 0.05 pmol each (from previous step) | BsaI-HFv2 or SapI | 1 | Molecular water | top up to 15 µL | Total | 15
For 7+ fragments (including backbone)
| Component | Volume (µL) |
--- | --- | | NEBridge Ligase Master Mix 3X | 10 | DNA fragments | 0.05 pmol each (from previous step) | BsaI or SapI | 1 if BsaI, 2 if SapI | Molecular water | top up to 30 µL | Total | 30
Run the appropriate cycle in the thermocycler:
2 fragment assembly (single insertion):
37°C for 15 min
60°C for 5 min
4°C infinite hold for retrieval
3-6 fragment assembly:
30 cycles of [37°C for 1 min + 16°C for 1 min]
60°C for 5 min
4°C infinite hold for retrieval
7+ fragment assembly:
30 cycles of [37°C for 5 min + 16°C for 5 min]
60°C for 5 min
4°C infinite hold for retrieval
Proceed to transformation or agarose gel electrophoresis, or freeze in -20°C for later use.
