The mutated oligonucleotide is used to direct the synthesis of the template, thereby altering the DNA sequence, with a mutation efficiency of 50-80%.
Operation method
basic program
Materials and Instruments
Escherichia coli Move 1. Transfer the phage spot produced by a single-stranded phage to a 1.5 ml microcentrifuge tube containing 1 ml of sterilized TY medium, incubate at 60°C for 5 min to kill the bacterial cells, shake vigorously to release the phage from the agar, and centrifuge for 2 min.2. Transfer 100 μl of supernatant to a 1 L flask containing 100 ml of medium containing 0.25 μg/ml of uridine. For more product details, please visit Aladdin Scientific website.
PEG TE ATP Oligonucleotide Mutagenesis Primer T4 Polynucleotide Kinase EDTA SSC
Water Bath Electrophoresis Instrument Incubator
3. Add 5 ml of E. coli culture medium in the middle of logarithmic growth and incubate at 37℃ for 6~18 hours with vigorous shaking.
4. Centrifuge at 5000 g for 30 min and retain the supernatant.
5. Determine the relative titer of phage in ung-E. coli to ung+ E. coli.
4. Add 1 volume of 5×PEG/NaCl solution to the supernatant of the 4-body Hoven to precipitate the phage, mix well and incubate at 0°C for 1 h.
6. Centrifuge at 5 000 g for 15 min, discard the supernatant, and dissolve the precipitate with 5 ml TE buffer in a 15 ml Corex tube with vigorous shaking on a vortex mixer.
7. Place the phage solution on ice for 1 h. Centrifuge again as in the previous step to remove cellular debris, then precipitate single-stranded phage DNA with phenol extraction and ethanol, and determine the DNA concentration by measuring the absorbance at 260 nm.8. Add the following reagents to a 1.5 ml microcentrifuge tube:
(1) 20 μl 10×T4 polynucleotide kinase buffer
(2) 2 μl 10 mmol/l ATP
(3) Mutated oligonucleotide (15~20 nucleotides long〉)
(4) Add water to 20 μl.
(5) 2 U T4 polynucleotide kinase
(6) The reaction was incubated at 37°C for 60 min, and the reaction was terminated by adding 3 μl of 100 mmol/l EDTA and heating to 70°C.9. Add a uracil-containing single-stranded circular DNA modifier (usually 1 μg DNA dissolved in 1 μl volume) to the phosphorylated oligonucleotide, add 1.25 μl of 20 × SSC, mix well and centrifuge for 5 s.
10. Place the centrifuge tube in a 500 ml beaker of 70 °C water, cool naturally to room temperature and centrifuge for 5 s on ice.
11. Add the following reagents to form a hybridization mixture:
(1) 20 μl 5× polymerase mixture
(2) 2.5 U or T4 DNA polymerase
(3) 2 U T4 DNA ligase
(4) Add water to 100 μl.
(5) were mixed thoroughly and then placed at 0°C for 5 min, room temperature for 5 min, and 37°C for 2 h. The results of the mixing were summarized as follows.
(6) Finally, add 3 μl 500 mmol/l EDTA to terminate the reaction.12. Electrophoresis 20 μl on 0.8% agarose. Control lanes should include single-stranded cyclic viral DNA, double-stranded closed-loop DNA, and double-stranded bad DNA with cuts.
13. Estimate the amount of DNA based on the electrophoresis results and transform ung+ E. coli with 1-100 ng of the double-stranded DNA product.
14. Selectively or randomly pick the resulting clones (phage or colonies) for isolation of pure clonal progeny.
15. The selected clones are analyzed by DNA sequencing.