Junction partitioning mutagenesis of DNA
Junction partitioning mutagenesis of DNA
Linker scanner mutations: In vitro additions of sites to restriction fragments that result in recombination of two DNA molecules, resulting in the addition of a linker sequence at the recombination site.
Operation method
Nested deletion and complementary oligonucleotides for splice partition mutagenesis
Materials and Instruments
DNA Move 1. Generate a nested 5' or 3-end' deletion mutant in the target region of the plasmid using Bal 31 or exonuclease III and S1 nuclease. First linearize the plasmid with a restriction enzyme in the vicinity of the target sequence, and when digesting with Bal 31, determine its cutting efficiency in order to find out how long it takes to cut 6-8 target base pairs and cover the region to be mutagenized.2. The synthetic junction is ligated to DNA that has been digested for an appropriate period of time. Cut with an appropriate restriction enzyme to produce a fragment with a junction at the end of the deletion and a vector site at the other end. Common Problems For more product details, please visit Aladdin Scientific website.
Mist Water Ethanol EDTA T4 DNA Ligase TE NaCl
Incubator Water Bath
3. The target fragment is separated on a low melting point agarose gel and ligated to the vector fragment to produce an intact plasmid with the deletion mutation.
4. Transform suitable E. coli with the ligation mixture, pick clones, and prepare plasmid DNA in small quantities.
5. Digest small quantities of prepared plasmid DNA with restriction endonucleases to produce small DNA fragments containing the deletion terminus at one end, and perform nondenaturing polyacrylamide or sieve agarose gel electrophoresis to determine the size of the DNA fragments as an indication of the presence of the deletion mutant terminus. Appropriate restriction enzymes are selected to obtain small fragments of 200-300 bp.
6. Sequence sets of deletion mutants within the target region to determine the exact deletion endpoints.
7. Design complementary oligonucleotide wafers to recover wild-type sequences located between the junction at each deletion end and the upstream restriction site of the 5' series of deletions, or between the junction and the downstream site of the 3' series of deletions.8. The following fragments were prepared for attachment to oligonucleotides:
(1) A segment cut from each deletion mutant that passes through the splice site and at a convenient site within the plasmid's drug resistance marker (pro-variant fragment).
(2) A backbone fragment cut from the wild-type plasmid that passes through the drug resistance site and a restriction site near the mutagenized region; isolate these fragments.9. Each oligonucleotide was resuspended at a concentration of approximately 100 μg/ml in TE buffer containing 15 mmol/l NaCl, and the various fragments were mixed in equimolar amounts and held at 65 °C for 10 min.
10. Slowly cool to room temperature for 20 min, measure A260 to determine the concentration, and monitor hybridization by electrophoresis of a small sample on a 4% sieved agarose gel.
11. join the three components as follows: mix the promoter and backbone fragments equimolarly, add 50 times the molar excess of hybridized oligonucleotides relative to the plasmid fragments, and the total amount of DNA is about 400 μl in 10 μl of total volume. add 0.4 μl of T4 DNA ligase, and incubate at 15°C for 2 h. If joining the promoter and backbone fragments by flat-end ligations, 1 μl of ligase should be added and incubated at 30°C for 2 h. If ligating by flat-end joining, add 1 μl of ligase. Ligase and hold at 30°C for 2 h. Dilute 40-fold to 1 μg/ml with TE buffer.
12. Inactivate the ligation reaction solution at 65°C for 10 min and cool slowly to room temperature.13. Transform suitable E. coli with 20 to 25 μl of diluted ligation reaction solution, spread on selection medium, 5% ligation solution produces 20 to 30 clones and all of them have the correct structure.
14. Pick clones, prepare plasmid DNA in small quantities, and verify the DNA by restriction enzyme digestion and DNA sequencing.
Figure I. Basic scheme generating joint zoning mutagenesis