DCLab: Gibson Assembly Designer

Multi-fragment overlap primer generation, plasmid mapping, and interactive cloning protocols.

1 Assembly Fragments (5' → 3' order)

Define fragments in the exact order they will assemble circularly.

2 Designed Primers

Fragment	Direction	Full Sequence (5' → 3')	Total Len	Bind Tm

Blue text indicates the overlap region (homologous to the adjacent fragment). Teal text indicates the template-binding region. Tm calculations apply only to the template-binding region for the initial PCR cycles.

3 Assembled Map

Total Size 0 bp

4 Interactive Cloning Protocol

Step A: PCR Master Mix

Calculate a shared master mix (lacking specific templates/primers) to aliquot into individual PCR tubes.

Rxn Volume: # Reactions: (+10% overage applied automatically)

Component	Per Rxn	Master Mix Total
5X HiFi Buffer (e.g. Q5/Phusion)	10.0 µL	--
10 mM dNTPs	1.0 µL	--
Polymerase	0.5 µL	--
Nuclease-Free Water	32.5 µL	--

Instructions: Aliquot 44.0 µL of the Master Mix into each PCR tube. Then, strictly to each individual tube, add:

2.5 µL of specific 10μM Forward Primer
2.5 µL of specific 10μM Reverse Primer
1.0 µL of specific Template DNA

*If template was a circular plasmid, treat final PCR product with 1 μL DpnI (37°C, 1 hr) before purification.

Step B: Gibson Assembly Reaction

Enter your purified PCR product concentrations (ng/µL) to automatically calculate volumes for a balanced 20 µL reaction.

Fragment	Len (bp)	Conc (ng/µL)	Target (pmol)	Add (µL)
Total DNA Volume:				--
Gibson Master Mix (2X):				10.00 µL
Nuclease-Free Water:				--

Incubate the 20 µL reaction at 50°C for 15-60 minutes.