Site Directed Mutagenesis Protocol

The protocols for three to five bases (50bp+ between two mutation sites) site-directed mutagenesis are similar. Below is the process for introducing 3 mutations:

Figure 1: Separate three base site-directed mutations
Choose the mutation sites A, B, and C as the boundary to divide the vector into fragments AB, BC, and CA. Design reverse complementary primers containing three mutation sites. Amplify fragment AB (A Forward Primer and B Reverse Primer), fragment BC (B Forward Primer and C Reverse Primer) and fragment CA (C Forward Primer and A Reverse Primer) with original plasmid as template. Amplification product treated by DpnI and used directly in the recombination reaction. Recombination product is transformed directly to complete the multiple base site-directed mutagenesis.

To introduce site-directed mutations of three separate nucleotides in the plasmid, three pairs of primers to amplify the plasmid in three parts are needed. The basic principles for primer design is as follows:
The 5’ ends of reverse and forward primers comprise 15-21 bp reverse complementary regions. 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 2 shows the detail of primer design, illustrated with the case of introducing three base mutations into vector pUC18.

Note: Primer design of primers at mutation sites B and C is the same as site A. 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.

The plasmid is divided in to fragments AB, BC and CA by mutation sites A, B, C. Use Genie Fusion Ultra High-Fidelity DNA Polymerase to amplify the three fragments. The primers to amplify AB are forward primer of mutation site A and reverse primer of mutation site B; The primers to amplify BC are forward primer of mutation site B and reverse primer of mutation site C. The primers to amplify CA are forward primer of mutation site C and reverse primer of mutation site A. 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 ℃ after use. The recommended reaction system is as follows:

a. Do not use dUTP, nor any primer and template containing uracil.
b. Given the normal amplification of the plasmid, use as less template as possible. Less than 1 ng of freshly extracted plasmid is recommended.
c. 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:

a. For most plasmids, the appropriate denaturation temperature is 95℃.
b. 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, the temperature gradient can be established to find the optimal temperature for primer binding to template. Too long annealing time may cause dispersed amplification products.
c. Long extension time can improve the yield of the amplification products.
d. In order to prevent the introduction of non-target mutation, 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 is subjected to gel electrophoresis. If the target plasmid is correctly amplified, please continue with the next step.

The amplification product of step 4.3 includes original template plasmid, so we need to digest the product with DpnI before recombination cyclization to prevent false-positive transformants after transformation. 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.

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. The following components are added sequentially to the bottom of a 1.5 ml sterile Eppendorf tubes or PCR tube on ice-water bath. If liquid adheres to the wall of the tube, collect the liquid to the bottom of tube by brief centrifugation.

The optimal amount of DNA fragments in the Exnase MultiS multi-base mutation recombination reaction system is 0.03 pmol of each fragment. The corresponding mass of DNA fragments can be calculated roughly by the following formula:

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

For example, if fragment AB is 1 kb, fragment BC is 2 kb, and fragment CA is 5 kb the optimal amount of fragment AB digested by DpnI is 20 ng (0.02× 1000 = 20 ng), fragment BC is 40 ng (0.02 × 2000 = 40ng), and fragment CA 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 components, please mix the components by gently pipetting up and down several times with a pipette, and avoid air bubbles (please do not vortex or shake vigorously). Incubate the tube at 37℃ for 30 min. After the reaction, please place the tube in ice bath for 5 min. The product can be transformed directly or stored at -20℃.

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 100μl of LB medium, and then plate all the bacterial cells.