GenieClone DNA Assembly Site-Directed Mutagenesis Kit v1

GenieClone Site-Directed Mutagenesis Kit v1 is designed for rapid site-directed mutagenesis of up to 2 separate mutations in the same reaction in less than 3 hours. Based on GenieClone rapid cloning technology, this kit uses homologous recombination to replace the conventional annealing ring-forming reactions meaning much less template and a more flexible primer design strategy. With seamless splicing of the two PCR products by GenieClone technology, the kit can complete up to two separate mutations with a single amplification reaction. DpnI digested products of the specific amplicons can be directly added to the recombination reaction without purification. The highly optimized reaction buffer, fast protocol and superior site-directed mutagenesis efficiency make GenieClone Site-Directed Mutagenesis Kit v1 make an excellent choice for introducing 1 or 2 mutations at discontinuous sites simultaneously on the same plasmid.

• Introduce up to 2 separate mutations in the same reaction.
• Site-directed mutagenesis can be performed on up to 2 discontinuous sites (more than 50 bp apart) simultaneously on the target plasmid.
• Single kit for cloning and mutagenesis.
• Includes Genie Fusion Ultra High-Fidelity DNA Polymerase providing high-fidelity PCR with extremely low mutation rates & excellent long fragment amplification for any plasmid amplification up to 20 kb as well as GC-rich templates.
• Amplification is carried out exponentially and the template usage is extremely low, which is beneficial to the complete degradation of the original methylation template.
• DpnI eliminates contamination of the original template and decreases background.
• Amplified products can be directly used in the recombination reaction after DpnI treatment.
• The reaction mixture can be used to directly transform chemically competent E.coli cells.

Note: use methylase non-defective host strains (eg. Top10, DH5α, JM109) to extract the original plasmids for use with this kit.

Store the product at -20°C, and it will be valid for 1 year

4.1 Overview of the experimental process (Figure 1)

1) Primer design (refer to 4.2);

2) Amplification of the target plasmid (Figure 1, I, refer to 4.3);

3) Amplification products digested by DpnI to remove the methylated template plasmid (Figure 1, I, refer to 4.4);

4) Recombination reaction (Figure 1, II, refer to 4.5);

5) Transformation with the recombination reaction products; plating of the transformants; Colony identification (Figure 1, III, refer to 4.6).

Figure 1: Single base site-directed mutation using GenieClone Site-Directed Mutagenesis Kit v1

4.2 Primer design

To introduce site-directed mutagenesis of a single base or continuous multiple, only one pair of primers for inverse amplification of the plasmid is required.

The basic principles for primer design are as follows:

The 5’ ends of reverse and forward primer comprise 15-21 bp reverse complementary region (GC content 40%-60%) and at least a 15 bp non-complementary region (the Tm value of the region between the mutation site and the 3’ end of the primer is recommended to be higher than 60℃). The mutation site can be in the complementary region (the mutation should be introduced om both primers) or in the non-complementary region of any one of the primers. The mutation site should not be at the end of the primer. Figure 2 details the primer design for introducing a single base mutation into vector pUC18.

Figure 2: Schematic of introducing single base or continuous multi-bases site-directed mutation into plasmid

Note: Calculation of the Tm value of primer should be based on the region between the mutation site to the 3 ' end of the primer. Tm value should exceed 60℃ by adjusting the length of primer. Please note that the region between the mutation site to the 5' end of the primer should not be included for calculation of the Tm value.

• Target Plasmid amplification

Use Genie Fusion Ultra High-Fidelity DNA Polymerase to amplify the target plasmid. Each component should be mixed well after thawing and placed back at -20℃ in time after use. The recommended reaction system is as follows:

1. Do not use dUTP, nor any primer and template containing uracil.
2. Given the normal amplification of the plasmid, use as little template as possible. Less than 1 ng of freshly extracted plasmid is recommended.
3. The recommended final concentration of enzyme is 1 U/50 μl. The optimal concentration of Genie Fusion Ultra High-Fidelity DNA Polymerase is 0.5 U to 2 U per 50 μl. No more than 2 U per 50 μl is recommended especially when the amplicon is longer than 5 kb.

After all the components are mixed, the recommended PCR program is as follows:

1. For most plasmids, the appropriate denaturation temperature is 95℃.
2. Genie Fusion Ultra High-Fidelity DNA Polymerase can promote the efficient annealing between the template and primers. In general, the annealing temperature is the Tm of primers. If required, the temperature gradient can be established to find the optimal temperature for primer binding to template. Excessive annealing time may cause dispersed amplification products.
3. Long extension time can improve the yield of the amplification products.
4. In order to prevent the introduction of non-target mutations, we recommend that the amplification cycle is less than 35 cycles. If the amplification efficiency is good, we recommend the amplification cycle number to be less than 30.

After the PCR reaction, a small amount of amplification product can be run via gel electrophoresis. If the target plasmid is correctly amplified, please continue with the next step.

• DpnI Treatment

The amplification product of step 4.3 includes original template plasmid, so DpnI treatment is required before recombination to prevent false-positives after transformation. The recommended reaction is as follows:

Place the reaction mixture at 37℃ for 1 to 2 hours. If the amplification product of 4.3 is single band, the DpnI digested products can be used in subsequent recombination reaction without purification. If the product is not single band, gel extraction purification should be performed before the next step.

• Recombination reaction

The 5’ ends of forward and reverse primers share a complete reverse complementary sequence, and thus homologous recombination can occur between 5’ end and 3’ end of the amplification product catalyzed by GenieClone Recombinase to complete the amplification product cyclization. Add the following components sequentially to the bottom of a 1.5 ml sterile Eppendorf tubes or PCR tube on ice. Briefly centrifuge the tube to bring all components to the bottom.

The optimal amount of DNA in the GenieClone Recombinase single-base mutation recombination reaction system is 0.03 pmol. The corresponding mass of DNA moles can be calculated roughly by the following formula:

The optimal amount of product digested by DpnI = [0.02 × the base pair number of target plasmid] ng (0.03 pmol)

For example, to introduce a single mutation into a 5 kb plasmid, the optimal amount of DpnI digestion product is 100 ng (0.02 × 5000 = 100 ng)

Note: Suboptimal DNA amounts will reduce the cyclization efficiency. Confirm the DNA concentration by gel electrophoresis in advance and mix the components strictly in accordance with the recommended amount. When the calculated optimal amount is less than 50 ng or more than 400 ng, then add 50 ng or 400 ng. When the product digested by DpnI is used directly in the recombination reaction, the volume of product should be less than 1/5 of the total volume.

After addition of all the components, mix the reaction system by gently pipetting up and down several times with a pipette and avoid air bubbles (do not vortex or shake vigorously). Incubate the tube at 37℃ for 30 min. After the incubation, place the tube on ice for 5 min. The product can be transformed directly or stored at -20℃.

• Transformation and colony identification

Add 20 μl of the cooled reaction mixture to 200 μl of competent cells. Mix gently by flicking the tube and place the tube on ice for 30 min. Heat shock the reaction by incubating the tube at 42℃ for 45 ~ 90 sec. Then incubate it on ice for 2 min. Add 900 μl of SOC or LB medium and incubate at 37℃ for 10 min.

Incubate by shaking (150 rpm) for 45 min at 37℃ and plate 100 μl of the bacterial culture on a selective plate. Incubate overnight at 37℃.

Note: We recommend competent cells whose transformation efficiency is higher than 10⁸ cfu/μg. If not, centrifuge your bacterial culture at 5,000 rpm for 3min to collect bacteria, resuspend with 100 μl of LB medium, and then plate all the bacterial cells.

1) Primer design (refer to 5.2);

2) Separate amplification of different segments of the target plasmid (Figure 3, I, refer to 5.3):

3) DpnI digestion of the amplification products to remove the methylated template plasmid (Figure 3, I, refer to 5.4);

4) Recombination reaction (Figure 3, II, refer to 5.5);

5) Transformation with the recombination reaction products; plating of the transformants; Colony identification (Figure 3, III, refer to 5.6).

Figure 3: Overview of separate double bases site-directed mutations with Mut Express II

4.7 Primer design

To introduce two separate site-directed mutations in the plasmid, two pairs of primers to amplify the plasmid in two parts are needed. The basic principles for primer design is as follows:

The 5’ ends of reverse and forward primers comprises 15-21 bp reverse complementary region. The mutation sites can be in the complementary region (the mutation should be introduced in both primers) or in the non-complementary region of any one of primers. The mutation site should NOT be at the end of the primer. Figure 4 shows the detail of primer design, illustrated with the case of introducing two base mutations into vector pUC18.

Figure 4: Schematic of primer design for introduction of separate double bases site-directed mutations

Note: Calculation of the Tm value of primer should be based on the region between the mutation site to the 3 ' end of the primer. Tm value should exceed 60℃ by adjusting the length of primer. Please note that the region between the mutation site to the 5 ' end of the primer should not be included for calculation of the Tm value.

• Target plasmid amplification

The plasmid is divided into fragment AB and fragment BA by mutation sites A and B. Use Genie Fusion Ultra High-Fidelity DNA Polymerase to amplify the fragments. The primers to amplify fragment AB are forward primer of mutation site A and reverse primer of mutation site B; The primers to amplify fragment BA are forward primer of mutation site B and reverse primer of mutation site A.

Each component should be mixed well after thawing and placed back at -20℃ each time after use. The recommended reaction system is as follows:

1. Do not use dUTP, nor any primer or template containing uracil.
2. Given the normal amplification of the plasmid, use as little template as possible. Less than 1 ng of freshly extracted plasmid is recommended.
3. The recommended final concentration of enzyme is 1 U/50 μl. The optimal concentration of Genie Fusion Ultra High-Fidelity DNA Polymerase is 0.5 U to 2 U per 50 μl. No more than 2 U per 50 μl is recommended especially when the amplicon is longer than 5 kb.

After all components are mixed, the recommended PCR program is as follows:

1. For most plasmids, the appropriate denaturation temperature is 95℃.
2. Genie Fusion Ultra High-Fidelity DNA Polymerase can promote the annealing between the template and primers efficiently. In general, the annealing temperature is the Tm of primers. If required, a temperature gradient can be established to find the optimal temperature for primer binding to template. Too long annealing time may cause dispersed amplification products.
3. Long extension time can improve the yield of the amplification products.
4. In order to prevent the introduction of non-target mutations, we recommend that the amplification cycle is less than 35 cycles. If the amplification efficiency is good, we recommend the amplification cycle to be less than 30.

After the PCR reaction, a small amount of amplification products are run via gel electrophoresis. If the target plasmid is correctly amplified, continue with the next step.

DpnI Treatment

The amplification product of step 4.3 includes original template plasmid. DpnI treatment before recombination cyclization is required to prevent false-positive transformants. The recommended reaction system is as follows:

Place the reaction mixture at 37℃ for 1 to 2 hours. If the amplification product of 4.3 is a single band, the DpnI digested products can be used in subsequent recombination reaction without purification. If the product is not a single band, gel extraction purification should be performed before the next step.

• Recombination reaction

The ends of fragments AB and BA share a region with exactly same sequence, and thus homologous recombination can occur between AB and BA catalyzed by GenieClone Recombinase to complete cyclization. The following components are added sequentially to the bottom of a 1.5 ml sterile Eppendorf tubes or PCR tube on ice. If liquid adheres to the wall of tube, collect the liquid at the bottom of tube by brief centrifugation.

The optimal amount of DNA fragments in the GenieClone Recombinase double-base mutation recombination reaction system is as follows: the long fragment is 0.03 pmol and the short fragment is 0.06 pmol. The corresponding mass of DNA fragments can be calculated roughly by the following formula:

The optimal amount of the long fragment digested by DpnI = [0.02 × the number of base pairs of fragment] ng (0.03 pmol)

The optimal amount of the short fragment digested by DpnI = [0.04 × the number of base pairs of fragment] ng (0.06 pmol)

For example, if fragment AB is 1 kb and fragment BA is 5 kb, the optimal amount of fragment AB digested by DpnI is 40 ng (0.04 × 1000 = 40 ng), and fragment BA is 100 ng (0.02 × 5000 = 100 ng)

Note: Suboptimal DNA amounts will reduce the cyclization efficiency. Confirm the DNA concentration by gel electrophoresis in advance, and mix the components strictly in accordance with the recommended amount. When the calculated optimal amount is less than 20 ng or more than 200 ng, then add 20 ng or 200 ng. When the product digested by DpnI is used directly in the recombination reaction, the volume of product should be less than 1/5 of the total volume.

After addition of all the components, mix the reaction system by gently pipetting up and down several times with a pipette and avoid air bubbles (do not vortex or shake vigorously). Incubate the tube at 37℃ for 30 min. After the incubation, place the tube on ice for 5 min. The product can be transformed directly or stored at -20℃.

Transformation and identification of clones

Add 20 μl of the cooled reaction mixture to 200 μl of competent cells. Mix gently by flicking the tube and place the tube on ice for 30 min. Heat shock the reaction by incubating the tube at 42℃ for 45 ~ 90 sec and incubate on ice for 2 min. Add 900 μl of SOC or LB medium and incubate at 37℃ for 10 min.

Incubate by shaking (150 rpm) for 45 min at 37℃ and plate 100 μl of the bacterial culture on a selective plate. Incubate overnight at 37℃.

Note: We recommend competent cells whose transformation efficiency is higher than 10⁸ cfu/μg. If not, centrifuge your bacterial culture at 5,000 rpm for 3min to collect bacteria, resuspend with 100 μl of LB medium, and then plate all the bacterial cells.

The following table lists main considerations when using this kit for site-directed mutagenesis (Table 1):

    Experimental procedure                                     Correct                                                         Incorrect

The following table lists main considerations when using this kit for site-directed mutagenesis (Table 1):

    Experimental procedure                                     Correct                                                         Incorrect