It's a snap! 9 considerations for easy multi-fragment cloning
The In-Fusion Cloning system is the highest-performing seamless cloning product on the market. It is particularly useful for more difficult cloning experiments, such as multiple fragment cloning (i.e., inserting more than one fragment simultaneously into a linearized vector) where the resulting colony number is far less compared to single fragment cloning. However, multi-fragment cloning requires specific considerations and careful experimental design to be successful.
Table of Contents
We provide answers for the most frequent questions about using the In-Fusion Cloning system for multi-fragment cloning. These tips and resources will help set up your experiment for success.
What are the advantages of seamless cloning compared to traditional methods for multi-fragment cloning?
Traditional cloning methods such as restriction-enzyme digestion followed by in vitro ligation are highly dependent on cloning site sequences within both the vector and the desired inserts. Other commonly used traditional cloning methods, such as TA cloning, require Taq polymerase, which is notorious for introducing errors during PCR. TA cloning is also non-directional, therefore inserting multiple fragments simultaneously with a specific direction and order is virtually impossible.
Seamless cloning, on the other hand, does not rely on the presence of restriction sites within a particular sequence. Users have complete control over what is inserted—and where—in the cloning reaction. This is particularly important when using multiple, unique DNA inserts. Additionally, In-Fusion Cloning follows a simple and elegant workflow, saving at least a day compared to traditional cloning workflows (Figure 1).
Figure 1. In-Fusion Cloning vs. traditional cloning workflows.
How is In-Fusion Snap Assembly different from other seamless cloning methods?
Both In-Fusion Snap Assembly and Gibson Assembly are seamless cloning methods; however, In-Fusion Cloning offers distinct advantages over Gibson Assembly and other seamless cloning methods. In-Fusion Cloning’s proprietary technology is a ligase-free and polymerase-independent seamless cloning method that introduces less background and errors, especially during challenging cloning experiments such as multi-fragment cloning. Additionally, the 15 min incubation time for In-Fusion Snap Assembly remains the same no matter how many fragments are inserted, unlike the Gibson Assembly method that requires up to one hour for a 4+ fragment assembly.
A performance comparison test using In-Fusion Snap Assembly and Gibson Assembly to create five-insert clones revealed that In-Fusion Snap yielded approximately 10 times more colony numbers as Gibson Assembly. Accuracy was ≥90% with In-Fusion Snap Assembly, but only 20% with Gibson Assembly (Figure 2). An at-a-glance multiple insert cloning protocol using In-Fusion Snap Assembly master mix is available.
Figure 2. Performance comparison between In-Fusion Snap Assembly and GeneArt Gibson Assembly HiFi when five fragments (405–1,005 bp) were cloned simultaneously into the pUC19 vector (2.7 kb).
What is the difference between In-Fusion Snap Assembly and In-Fusion HD?
Compared to In-Fusion HD, In-Fusion Snap Assembly’s improved formulation yields more colonies while retaining accuracies of ≥95%. In-Fusion Snap Assembly follows the same workflow as In-Fusion HD with improved cloning performance, particularly for challenging cloning experiments.
Is In-Fusion Snap Assembly EcoDry Master Mix suitable for multi-fragment cloning?
Yes. The In-Fusion Snap Assembly EcoDry Master Mix, provided in a convenient, lyophilized format, comes in either an 8-tube strip or 96-well plate and yields high cloning efficiency, especially for large or multiple fragments. An at-a-glance protocol using In-Fusion Snap Assembly EcoDry Master Mix for multiple insert cloning is available.
Do I need to choose a specific In-Fusion kit for multi-fragment cloning?
No. In-Fusion Cloning is very versatile; you can use the same kit for all your experiments, whether you are performing single- or multiple-fragment cloning or using small or large inserts.
Is the protocol for In-Fusion multi-fragment cloning different from single-insert protocols?
The overall workflow is the same for In-Fusion multi-fragment cloning and single-insert cloning with two exceptions for multi-fragment cloning:
- Use longer homologous sequences for multi-fragment cloning. A major difference between multi-fragment and single-insert cloning is the length of overlapping sequences between adjacent fragments. In-Fusion Cloning usually requires a 15 bp overlap, but we recommend using a 20-bp overlap for multi-fragment assembly to increase specificity.
- Use Stellar competent cells. Multi-fragment cloning requires a synergistic effect on assembly and transformation reactions. Stellar competent cells are optimized to work synergistically with In-Fusion Snap Assembly.
How do I design In-Fusion primers for multi-fragment assembly?
Designing primers to amplify each insert is easy using the online Primer Design Tool, which takes into account an increased overlapping region when using multiple inserts to increase cloning efficiency. To add more inserts, click ‘Add another insert’ (red arrow in Figure 3 below) in the tool.
Figure 3. Using the Primer Design tool to design primers for multiple inserts.
Use the following tips when designing primers to ensure successful cloning experiments:
- Each PCR primer for multi-insert cloning must be designed to generate products containing 5’ ends with 20 bp of homology to the ends of the linearized vector or other inserts.
- Avoid complementarity within each primer and between primer pairs
To learn more, refer to the In-Fusion Cloning primer design tutorial page.
How do I determine the molar ratio for multi-fragment assembly?
Generally, the molar ratio of each insert should be 2:1 to the linearized vector (i.e., two moles of each insert for each mole of linearized vector). For example, the molar ratio of two inserts with one vector will be 2:2:1. Good cloning efficiency is achieved when using a combined 200 ng of vector and inserts at this molar ratio in a 10 μl reaction. The molar ratio calculator allows users to calculate the molar ratio of up to 5 inserts.
Troubleshooting
Fewer colonies than expected
- Run the In-Fusion positive control reaction. Successful assembly with the positive control indicates that the experimental system is functioning as expected and issues may be specific to your vector and/or inserts.
- Ensure 20 bp overlap sequences between adjacent fragments. The longer overlap guarantees increased specificity in multi-fragment cloning.
- If using another type of competent cells, consider trying Stellar competent cells, which are optimized to work synergistically with In-Fusion Snap Assembly. We highly recommend using Stellar competent cells, particularly for multi-fragment cloning experiments, which typically yield fewer colonies than single fragment cloning protocols. Takara Bio offers convenient In-Fusion Snap Assembly Master Mix and Stellar competent cell bundles.
- The volume of the transformation reaction plated must be high enough to yield an adequate number of colonies. We recommend plating 1/100–1/5 of each transformation reaction diluted with SOC medium up to 100 µl for most reactions. For multi-fragment cloning, it may be necessary to plate a larger volume (1/5–1/3 of each transformation reaction) to ensure sufficient colony numbers.
No insert and/or wrong inserts
- No insert can be due to an undigested vector. If the vector size is ≥8 kb, restriction enzymes should be used to linearize the vector, since PCR amplified vector is not easily differentiated from template DNA on gel electrophoresis. Make sure you remove any uncut or undigested vector prior to use in the In-Fusion reaction. If necessary, recut the digested vector and gel purify prior to use.
- If your colony contains the wrong insert, check whether each PCR product yields a single band at the expected size using gel electrophoresis. If the PCR product has non-specific bands, it is necessary to gel purify the PCR product to ensure cloning of the correct insert(s).
Resources
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