Region-specific mutagenesis experiments
Region-specific mutagenesis experiments
Region-specific mutagenesis experiments for (1) gene-induced modification (2) specific expression of genes (3) clinical anti-tumor and other targets.
Operation method
region-specific mutagenesis
Principle
A large number of randomly distributed nucleotide substitution mutations can be generated on clonized DNA fragments up to 3' long, which are treated with a variety of chemical reagents that can cause damage to specific bases after the DNA has been cloned into a vector that can be used to isolate single-stranded DNA.
Materials and Instruments
DNA Move 1. Digest the DNA with restriction endonuclease to obtain the target DNA fragment and bi-directionally clone the target fragment into M13 phage vector or plasmid vector containing M13 replication initiator. Single-stranded DNA was extracted from 100 ml of infected fine-holding cultures. Caveat Since the performance of different chemical mutagens varies greatly, it is necessary to do a clear analysis of the temperature, PH value, light, solvents, etc., which affect their effects, and at the same time, their half-life, toxicity and protection should also be fully understood, so as to ensure the effectiveness of the use of the use of the effectiveness of the human body and safety. Common Problems 1. Due to the great difference in the performance of different chemical mutagens, before using them, we must do a clear analysis of the temperature, PH value, light, solvents, etc., which affects their effects, and at the same time, we should fully understand their half-life, toxicity and protection, so as to ensure the effectiveness of the use of the human body and safety. 2. When the credibility of the screening method is high, often the detection method is more cumbersome (especially involving some protein or amino acid content changes in the type of mutation) in order to improve the screening workload tends to reduce the credibility of the screening to reduce the degree of cumbersome screening, but in the re-screening to further determine. For more product details, please visit Aladdin Scientific website.
Sodium acetate Sodium nitrite TE Anhydrous ethanol dNTP Reverse transcriptase RNAase Elution Ethidium bromide
Water bath Incubator
2. 40 ug 1 mg/ml of single-stranded DNA is added to each of three microcentrifuge tubes, six reactions are used if both strands of the target sequence are to be utilized.3. nitrite reaction: add 10 ul 2.5 mol/l sodium acetate at pH 4.3 and 50 ul 2 mol/l sodium nitrite in one tube, mix well and incubate for 60 min at room temperature.
4. Formic acid reaction: add 60 ul concentrated formic acid into another tube, mix well and incubate at room temperature for 10 min.
5. Hydrazine reaction: Add 60 ul of concentrated hydrazine to a third tube, mix well and incubate at room temperature for 10 min. 6.
6. To each tube, add 100 ul of 2.5 mol/l pH 5.5 sodium acetate, 30 ug of tRNA and 1 ml of anhydrous ethanol, freeze in dry ice at -80°C for 10 min, centrifuge for 10 min, remove supernatant, repeat the ethanol precipitation twice, and then resuspend the DNA in 80 ul of TE buffer. 7.
7. Add 10 ul of 4 kinds of dNTP mixture, 10 ul of 10x reverse transcriptase buffer and 100 pmol of oligonucleotide primer to the resuspended DNA. First incubate at 85 ℃ for 5 min, and then incubate at 40 ℃ for 15 min. 8.
8. Add 10 ul 4 kinds of dNTP mixture and 30~40 U of AMV reverse transcriptase, and incubate at 37 ℃ for 1 h.
9. Equilibrate phenol extraction and ethanol precipitation of DNA with buffer, resuspend DNA in 100 ul 10x restriction endonuclease buffer and cut with appropriate restriction endonuclease to recover fragments from the vector.
10. phenol extraction and ethanol precipitation of DNA. the DNA was resuspended in 10-15 ul elution buffer with 0.5 ul 2 mg/ml RNAase A and incubated at 37°C for 15 min to degrade the stretcher tRNA.11. Separate the target fragment cut from the vector on a nondenaturing 6% polyacrylamide gel electrophoresis, the gel was stained with ethidium bromide, the gel was cut, and the DNA was eluted.
12. Ligate the purified insert DNA into a plasmid or bacteriophage vector with complementary ends. The ligation mixture is introduced into suitable bacteria by conventional transformation procedures.