Assembly¶
In molecular cloning, “assembly” refers to joining two or more DNA molecules together into one. Two of the most common methods are Golden Gate Assembly and Gibson Assembly—together they cover nearly all modern cloning needs.
Assembly Methods Overview¶
Golden Gate Assembly uses a type IIS restriction enzyme (e.g., BsaI, BsmBI, BbsI) to cut DNA at defined locations and generate sticky ends. These are then joined by T4 DNA Ligase.
Gibson Assembly uses an exonuclease to chew back DNA ends, allowing single-stranded overlaps to anneal. A polymerase fills in gaps and a ligase seals the nicks.
Both methods:
- Are performed in a single tube (one-pot)
- Run in a thermocycler
- Require specific enzyme buffers and careful setup
What We're Doing Here¶
In the pP6 experiment, we only have one DNA fragment: the linear PCR product from earlier. Our goal is to re-circularize it by joining its ends together using Golden Gate.
This works because the PCR primers added BsaI recognition sites, which generate complementary sticky ends at each end of the linear product.
Golden Gate Diagram¶
Figure: Schematic of the pP6 assembly process. The plasmid pJ12 is first amplified by PCR using primers that introduce BsaI sites and overhangs. The resulting linear product is then re-circularized via Golden Gate Assembly. This is a special case of Golden Gate Assembly, also referred to as enzymatic inverse PCR (EIPCR), where the input and output are the same plasmid sequence in linear and circular forms, respectively.
In the pP6 experiment, we only have a single linear DNA molecule—the full plasmid amplified by PCR. The primers introduce BsaI sites at both ends. When digested, the ends form compatible overhangs that allow the DNA to ligate back into a circle. This is a special case of Golden Gate where the product is a re-circularized version of the input.
Figure: The linear PCR product is digested by BsaI to expose sticky ends. These are self-compatible and ligate to form a circular plasmid.
Reaction Setup¶
Setting up a Golden Gate reaction is similar to setting up PCR. You pipette reagents in order from top to bottom, adding enzymes last to avoid denaturing them in pure water or incomplete mixtures.
| Volume | Reagent | Tube Label |
|---|---|---|
| 6 µL | ddH₂O (white rack) | W____ |
| 1 µL | 10× T4 Ligase Buffer (red) | L____ |
| 2 µL | z79 (your cleaned PCR DNA) | |
| 0.5 µL | T4 DNA Ligase | |
| 0.5 µL | BsaI |
- Label your reaction tube with your assigned code (e.g.,
a79). - Place all reactions from your section together in a single thermocycler block.
Thermocycler Program¶
We use the program GG1, which alternates between:
- 37°C (cutting by BsaI)
- 16°C (ligation by T4 Ligase)
The final steps:
- Extended 37°C incubation
- 65°C heat inactivation
Lab Sheet Overview¶
| Label | Fragment | Product |
|---|---|---|
| a79 | P6 | pP6 |
The Label is what you write on your PCR tube. P6 refers to the PCR product you're assembling. Once the reaction is complete, the product is a circularized plasmid called pP6, ready for transformation.
Scaling the Reaction¶
You can scale this up or down depending on your needs, but:
- Final buffer concentration must be 1×
- Avoid excessive DNA concentrations that may inhibit ligation
Typical scale is 10 µL, matching what’s needed for competent cell transformation.