In this method, two oligonucleotide primers hybridize to the same strand of the DNA duplex of a denatured recombinant plasmid. One primer (mutagenic primer) carries a mutation that is intended to be introduced into the target DNA sequence, and the second primer carries a mutation that disrupts a single restriction enzyme site on the plasmid. This experiment was derived from the next volume of the Molecular Cloning Laboratory Guide (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
USE mutation experiment
Materials and Instruments
Transformed E. coli sensory state with mutS phenotype (e.g., BMH71-18) Transformed E. coli sensory state with mut+ phenotype Move makings For more product details, please visit Aladdin Scientific website.
Annealing buffer Storage solution Synthesis buffer Phage T4DNA ligase Phage T4DNA polymerase or sequencing enzyme Single site restriction endonuclease Agarose gel Mutagenesis primer Selection primer Plasmid DNA
70°C water bath and water bath suitable for the temperature of the restriction enzyme digestion reaction LB agar plates and LB liquid medium with suitable antibiotics
Buffers and solutions
Refer to Appendix 1 for the composition of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.
10x Annealing Buffer
200 mmol/L Tris-Cl (pH 7.5)
100 mmol/LMgCl2
500 mmol/LNaCl
10X synthesis buffer
100 mmol/L Tris-Cl (pH 7-5)
dTTP, dATP, dCTP, dGTP 5 mmol/L each
10 mmol/LATP
20 mmol/LDTT
Enzymes and buffers
Phage T4DNA Ligase
Phage T4DNA polymerase or sequencing enzyme
Phage T4 encodes a natural DNA polymerase that cannot displace oligonucleotide primers from template DNA (Nossal 1974. Kunkel 1985, Bebenek and Kunkel 1989, Schens 1989).
A single-site restriction endonuclease
Gel
1% agarose gel (containing 0.5ug/ml ethidium bromide)
Nucleic acids and oligonucleotides
Mutagenic primers
Selection primers
Mutagenic primers and selection primers must anneal to the same strand of the target DNA, and the 5' end of each primer must be phosphorylated. Mutagenesis primer design is described in the information section on Mutagenesis Oligonucleotides. The mutation should be located in the middle of the primer and the mutation site should be flanked by 10-15 bases that pair exactly with the template DNA. Oligonucleotide primers are purified by high-pressure liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE) prior to use (see Chapter 10, Protocols 1 or 5). Several companies (Clomech; Pharmacia) sell selection primers, and some (e.g., Pharmacia) also sell pairs of "toggle" primers, which can be used to restrict forward or reverse transitions of the locus, allowing for multiple rounds of ordered mutations. No subcloning template is required.
Plasmid DNA
Closed and looped plasmid DNA can be purified by column chromatography or base lysis with commercially available resins (see Chapter 1, Protocols 2 or 9).
Culture media
LB agar plates and LB liquid medium with appropriate antibiotics
Specialized equipment
70°C water baths and water baths suitable for the temperature of restriction enzyme digestion reactions
Other reagents
The reagents required for steps 7 and 14 of this protocol are listed in Chapter 1, Protocols 23, 24, or 26, and the E. coli transformation is described in Protocol 26.
The reagents required for the small amounts of plasmid DNA prepared in steps 10 and 16 of this protocol are listed in Chapter 1, Scheme 1.
The reagents required for step 17 of this protocol are listed in Chapter 12, Schemes 3, 4, and 5.
Vectors and Strains
See Appendix 3
E. coli receptor for transformation with mutS phenotype (e.g., BMH71-18)
Transformed E. coli sensors with mut+ phenotype
See step 14.
Methods
Synthesis of mutant DNA strands
1. Mix the following in a microcentrifuge tube.
10x annealing buffer 2ul
Plasmid DNA 0.025 to 0.25 pmole
Selection primers 25pmoles
Mutation primers 25pmoles
Add water to 20ul
Incubate in boiling water for 5 min.
The amount of plasmid DNA used depends on the primers and plasmid DNA used in the reaction.
2. Immediately cool the centrifuge tube on ice for 5 min. centrifuge for 5 s in a microcentrifuge to concentrate the liquid at the bottom of the tube.
3. Add the following components to the centrifuge tube containing the annealed primer and plasmid DNA:
10X Synthetic Buffer 3ul
Phage T4DNA Polymerase (2-4 units/ul) 1ul
Phage T4DNA Ligase (4~6 units/ul) 1ul
Water 5ul
Mix the reagents with a pipette by gently blowing up and down several times. Incubate the reaction at 37°C for 1-2 h. Centrifuge in a microcentrifuge for 5S to concentrate the liquid at the bottom of the tube.
4. Heat the tube at 70°C for at least 5 min to inactivate the enzyme and terminate the reaction. Place the tubes on a bench to cool to room temperature.
Primary screening by restriction endonuclease digestion
5. Adjust the concentration of NaCl to a concentration suitable for a single site restriction enzyme reaction. Use 10X annealing buffer,NaCl storage solution, or the 10x buffer provided with the restriction enzyme.
In a total volume of 30ul of synthesis or ligation mixture, the NaCl concentration is 37.5 mmol/L. If NaCl is not required for the restriction digestion reaction, or if NaCl is required at a lower concentration than that described above, the DNA mixture in the synthesis and ligation buffer can be precipitated with ethanol or passed through a centrifugal column suspended in the appropriate restriction enzyme buffer.
6. 20 units of restriction endonuclease of choice are added to the reaction mixture. Digest for at least 1 h at a suitable temperature.
IMPORTANT: The volume of enzyme in the reaction (including polymerase and ligase) should not exceed 10% of the total reaction volume. The reaction volume should be adjusted accordingly to the above ratio.
Round 1 Transformation and Enrichment of Mutant Plasmids
7. Transform a mutS E. coli strain such as BMH71-18 with the plasmid DNA contained in the digestion mixture according to the transformation procedure described in Chapter 1, Schemes 23-26.
8. Spread LB agar plates containing appropriate antibiotics with 10, 50 and 250ul of the transformation mixture. Incubate the dishes at 37°C overnight. Perform step 9 during incubation of Petri dishes.
These dishes were used to determine the number of transformants. Each 50ul of transformation mixture coated dish should yield 100-300 clones.
9. Add 3 ml of LB culture solution with appropriate antibiotics to the remaining transformation mixture to amplify the plasmids. incubate overnight at 37°C with shaking.
10. Prepare plasmid DNA the next day from approximately 2.5 ml of the overnight culture (see Scheme 1 in Chapter 1).
11. Digest the plasmid DNA prepared in step 10 with the restriction enzyme of choice.
Plasmid DNA 500ng
10x Restriction Enzyme Buffer 2ul
Single-site restriction endonuclease 20ul
Add water to 20ul
Incubate the reaction for 2 h at the appropriate temperature.
12. Add 10 units of restriction enzyme and incubate for at least 1h.
13. Take 5-10ul of plasmid DNA and electrophoresis it on 1% agarose gel (containing 0.5ug/ml ethidium bromide) and evaluate the digestion result by gel electrophoresis.
The undigested (looped) DNA was separated by gel electrophoresis into two bands corresponding to the relaxed looped or superhelical form of DNA, respectively. linearized plasmid DNA showed different bands from the looped DNA. However, since the parental plasmid makes up the majority of the total plasmid DNA, the band corresponding to the undigested mutant plasmid DNA may be quite weak compared to the band of the digested parental plasmid DNA.
Final round of transformation
14. Take 2~4ul of digested plasmid DNA (about 50~100ng) and transform a chemically treated E. coli mutS+ strain, or transform an E. coli mutS+ strain by electroshocking with a 5-fold dilution of 1ul of plasmid DNA in sterilized water (about 5 ng) (see protocols 23~26 in Chapter 1).
15. 10, 50 and 250ul of the transformation mixture should be spread on LB agar dishes containing appropriate antibiotics. incubate the dishes at 37°C overnight.
16. On day 2, at least 12 individual transformants are picked for small-volume plasmid DNA preparation, digested with restriction endonuclease, and identified by agarose gel electrophoresis for plasmids resistant to selective restriction endonuclease digestion.
17. DNA sequence analysis is performed to identify plasmids containing the intended mutation (see Protocols 3, 4, and 5 in Chapter 12). 