1. Think about your final construct. Choose the vector you want to modify and envision your final, mutated construct (Figure 1; mutation shown in yellow).
2. Design your primers. Design inverse primers that overlap each other by 15 bp at their 5' ends and incorporate your desired deletion, substitution, or addition. Specific guidelines for mutagenesis primer design are described below.
3. Utilize the power of In-Fusion technology. Using an inverse PCR protocol, amplify the vector with your new primers. Perform the In-Fusion Cloning reaction using the PCR product. The linear DNA will re-circularize at the site of the 15-bp overlap and will also contain your mutagenic changes. Use a portion of the In-Fusion Cloning reaction to transform the Stellar Competent Cells according to the In-Fusion Snap Assembly protocol.
4. Obtain your final construct. Recover your mutant from the Stellar cells the following day.

Figure 1. Procedure for performing mutagenesis with In-Fusion technology. The area where mutagenesis occurs is shown in yellow. After designing your experiment, perform the protocol (Step 3 above) on Day 1, and recover your final construct (Step 4 above) on Day 2. Note: Although all examples shown here involve protein coding (gene) sequences, you can use the same methods to modify noncoding sequences such as promoters or transcription factors.

Primer design is a key component of simple deletion mutagenesis using In-Fusion technology. To delete a region of your cloning vector, you must design primers that include 15-bp overlaps with each other at their 5' ends and do not include the bases to be deleted (Figure 2).

For easy understanding of the primer design concept, different regions of the vector backbone and primers are marked in color in the figure below. The deletion site is marked in yellow and the binding site for the reverse primer (pink and turquoise) spans the deletion. The binding site for the forward primer (turquoise and black) is located against the cloning vector backbone. The two primers overlap by 15 bp at their 5' ends (the common area in turquoise). Note that there is no gap between the pink and turquoise regions in the actual primer sequence—the deleted nucleotides are not included in either of the primers.

Figure 2. In-Fusion primer design for deletion mutagenesis. Primers are designed to eliminate a section of the original vector.

To create a series of C-terminal deletions, design only one forward primer that anneals to your cloning vector immediately downstream of the coding region, retaining the stop codon. Then design a series of reverse primers that include 15 bp of overlap with the forward primer at their 5' ends and span different regions to be deleted. In Figure 3, Construct A has the blue region deleted, Construct B has the blue and turquoise regions deleted, and Construct C has the blue, turquoise, and pink regions deleted.

Figure 3. In-Fusion primer design to create a series of C-terminal truncated proteins. Primers are designed to eliminate one or more contiguous sections of the original vector.

To create a series of N-terminal deletions, design one reverse primer that anneals to your cloning vector immediately upstream of the coding region, including a start codon. Then design a series of forward primers that retain the natural start codon, include 15 bp of overlap with a reverse primer at their 5' ends, anneal to the coding region you wish to maintain at their 3' ends, and span different deletions. In Figure 4, Construct D has the turquoise region deleted and Construct E has the turquoise and blue regions deleted.

Figure 4. In-Fusion primer design to create a series of N-terminal truncated proteins. Primers are designed to eliminate one or more contiguous sections of the original vector.

To insert bases with In-Fusion Cloning technology, simply design primers that include 15-bp overlaps with each other at their 5' ends and contain the desired insertion(s) within the overlapping region (Figure 5). Only the 15 bases at the 5' ends of the primers are required to overlap, but depending on the length and sequence of your insertion, the overlap may be longer than 15 bp. Additional bases added to the primer will be maintained after the In-Fusion Cloning reaction.

Similarly, if you would like to change one or more bases in a construct, design primers that include 15-bp overlaps with each other and contain the desired substitutions within the overlapping region (Figure 5).

Figure 5. In-Fusion primer design for inserting or substituting bases. Primers are designed to insert additional bases or replace existing bases with different nucleotides (shown as Xs in the figure) in the original vector.

Please see the In-Fusion Snap Assembly user manual for detailed instructions.

1. Select your vector and identify the mutation site.
2. Design PCR primers as described above, keeping in mind these general guidelines:
   - Primers should be 18–25 bases long. Insertions may require longer primers.
   - Primers should be 40–60% GC.
   - Primer T_ms should be 58–65°C. The difference between forward and reverse primer T_ms should be ≤4°C.
3. Prepare PrimeSTAR Max DNA Polymerase master mix:
   

   PrimeSTAR Max DNA Polymerase	12.5 μl
   Forward primer	200–300 nM
   Reverse primer	200–300 nM
   Template	0.1–5.0 ng
   H2O	As needed
   Total reaction volume	25 μl

4. Linearize the vector by inverse PCR using a three-step PCR protocol and PrimeStar Max DNA Polymerase.
   

   98°C	10 sec
   55°C	5 or 15 sec		30–35 cycles
   72°C	5 sec/kb

5. Gel-purify the PCR product using the NucleoSpin Gel and PCR Clean-Up kit.
6. Assemble the In-Fusion Cloning reaction:
   

   Linear construct containing your mutation	100 ng
   In-Fusion Snap Assembly Master Mix	2 μl
   H2O	As needed
   Total reaction volume	10 μl

7. Incubate the reaction at 50°C for 15 min.
8. Transform Stellar Competent Cells with 2.5 μl of the In-Fusion Cloning reaction.
9. The next day, screen for mutants. You have a ≥95% chance of recovering your final desired construct the very first time.

Ochman H., Gerber, A. S., Hartl D. L. Genetic applications of an inverse polymerase chain reaction. Genetics 120(3):621–623 (1988).