Mutagenesis PCR experiment
Mutagenesis PCR experiment
The greatest advantage of mutagenic PCR is that various types of mutations are introduced in a preference-free manner, rather than obtaining a high level of single amplification. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Mutagenesis PCR experiment
Materials and Instruments
Chloroform Isoamyl alcohol Ethanol Mineral oil or a wax bead 3 mol L NaOAc 5 mmol L MnCl2 Mutant PCR buffer DNA polymerase Native Taq or AmpliTaq DNA polymerase High purity deoxynucleoside 5'-triphosphate dNTP mixture Template DNA primers Agarose gel stained with ethidium bromide Move I. Materials For more product details, please visit Aladdin Scientific website.
Thermocycler
1. Buffers, solutions and reagents
Chloroform/isoamyl alcohol (24:1, v/v)
Ethanol, 100%
Mineral oil or a wax bead
3mol/L NaOAc,pH5.2
5 mmol/L MnCl2
1X Mutation PCR buffer
70 mmol/L MgCl2
500 mmol/L KCl
100 mmol/L Tris-HCl, pH 8.3 at 25°C
1g/L gelatin
2. Enzyme and enzyme buffer
DNA polymerase
Native Taq or AmpliTaq DNA polymerase (Applied Biosystems or other licensed vendors do not substitute other heat-stable DNA polymerases)
3. Nucleic acids and oligonucleotides
High purity deoxyriboside 5'-triphosphate (Pharmacia, USB/Amersham)
10XdNTP mixture containing 2 mmol/LdGTP, 2 mmol/LdATP, 10 mmol/LdCTP and 10 mmol/LdTTP
Template DNA
Primers
4. gels
Agarose gel stained with ethidium bromide
5. Special equipment
Thermocycler
II. Methods
1. Prepare 10X mutant PCR buffer.
2. Prepare 10X dNTP mixture.
3. Prepare a 5 mmol/LMnCl2 solution, do not mix with the 10X PCR buffer or a precipitate will be generated that will interfere with PCR amplification.
4. Mix 10ul of 10X Mutation PCR Buffer, 10ul of lOXdNTP mixture, 30pmol of each primer, and 20fmol of template DNA together, make up the total volume to 88ul with H20, and mix.
5. Add 10ul of 5 mmol/L MnCl2 solution and mix well to ensure no precipitate forms.
6. Add 5U (2ul) of Taq DNA Polymerase to bring the final volume to 100ul and mix gently. Cover with mineral oil or a wax bead if desired.
7. Incubate for 30 cycles at 94°C for 1 min, 45°C for 1 min, and 72°C for lmin. Do not set a hot start step or an extended extension time at the end of the last cycle.
8. Purify the reaction product by first extracting with chloroform/isoamyl alcohol (24:1, v/v) followed by precipitation with 0.25 mol/LNaOAc and 2.5x 100% ethanol.
9. A small portion of the purified product is assayed in an agarose gel and the amount of full-length product is confirmed to be satisfactory by ethidium bromide staining. The mutant PCR should be performed in parallel with the standard PCR (ignoring the 4 differences listed above) and the amount of full-length DNA obtained by both PCRs should be similar.