PCR mutation experiments by overlap extension and gene SOEing
PCR mutation experiments by overlap extension and gene SOEing
This experiment describes about the process of PCR mutation by overlap extension and gene SOEing. This experiment is from PCR Lab Guide (2nd edition) by Seed Kang and Qu Lijia.
Operation method
PCR mutation experiments by overlap extension and gene SOEing
Materials and Instruments
Sterile H2O PCR buffer Restriction enzymes and buffers Taq DNA polymerase PCR templates 5' and 3' primers dNTP Move I. MATERIALS For more product details, please visit Aladdin Scientific website.
DNA sequencing device Thermocycler Agarose gel electrophoresis device
1. Buffers, solutions and reagents
Sterile H2O
2. Enzymes and enzyme buffers
10X PCR buffer (Roche) containing 100 mmol/L Tris-HCl, pH 8.3, 500 mmol/L, KCl and 15 mml/L MgCl2
Restriction enzymes and buffers
Taq DNA polymerase (Roche)
3. Nucleic acids and oligonucleotides
10X dNTP, commercially available dNTP solution (Amersham Biosciences) is a 100umol/L stock solution that can be diluted with sterile water.
PCR templates
5' and 3' primers
4. Special equipment
DNA sequencing device
Thermocycler
5. Other
Agarose, the concentration of agarose required to isolate DNA fragments depends on the size of the fragments to be isolated. size of the fragments to be isolated. As a rule of thumb, 0.8% to 1% SeaKem GTG agarose (Cambrex) (prepared with 1XTAE) is generally satisfactory for separating DNA fragments of 0.25 to 2kb in size.
Agarose gel electrophoresis equipment
GENECLEAN II kit (BIO101)
6. Vectors and strains
Cloning vectors and strains for sequencing of PCR products.
METHODS
1. PCR Mutation by Overlap Extension
(1) PCR reactions are performed in separate microcentrifuge tubes to generate the products AB and CD (see Figure 32-1). These reaction mixtures can be mixed at room temperature. 
(2) Perform 30 cycles of the reaction (94°C for 1 min, 50°C for 1 min, 72°C for 2 min).
(3) All reactants from each reaction were subjected to agarose gel electrophoresis.
(4) The target bands (i.e. products AB and CD) were cut off and the DNA fragments were recovered using the GENECLEANU II kit.
(5) Perform the overlap extension reaction in a new microcentrifuge tube. 
This reaction is not very sensitive to the amount of template added. Usually 10-100ng of each template can be used. primers a and d can also be added at the beginning of the PCR reaction.
(6) Perform PCR amplification as in step 2 above.
(7) Perform agarose gel electrophoresis of the reaction, cut off the target band (i.e., product AD), and recover the DNA fragments as described in step 4.
(8) The purified mutant product AD can then be digested by an appropriate restriction enzyme and ligated into a suitable cloning vector for subsequent transformation and sequencing.
2. Gene SOEing
(1) Perform the PCR reaction in separate microcentrifuge tubes to produce the product WX from gene 1 and the product YZ from gene 2, respectively (Figure 32-2). 
(2) The reaction is performed for 30 cycles (94°C for 1 min, 50°C for 1 min, 72°C for 2 min).
(3) All reactants from each reaction were subjected to agarose gel electrophoresis.
(4) The target bands (i.e., products WX and YZ) were cut off, and the DNA fragments were recovered using the GENECLEANB kit.
(5) Perform the SOEing reaction in a microcentrifuge tube. 
(6) Perform PCR amplification as described in Step 2 above.
(7) Perform agarose gel electrophoresis of the reaction, cut off the target band (i.e., product WZ), and recover the DNA fragments as described in Step 4.
(8) The purified mutant product WZ can then be digested by appropriate restriction enzymes and ligated into a suitable cloning vector for subsequent transformation and sequencing.