Muta-Direct™ Site Directed Mutagenesis Kit
Products for mutation induction and mutagenesis in three-step shift position
The Muta-Direct™ Site-Directed Mutagenesis Kit is a product used to induce mutations in the desired region of the gene cloned in the plasmid DNA. The Muta-Direct™ Site-Directed Mutagenesis Kit shows the mutation induction efficiency of 100% in theory. It is a very convenient and convenient product to finish the stage. It is a product designed to specifically induce mutation of the desired site without using special vector such as M13 or DNA methylastation step. Site-directed mutagenesis refers to the change of a specific DNA sequence. It can also be used to identify functional sites (catalytic domains) by studying the function of a protein by replacing, inserting, or removing amino acids at specific sites with other amino acids and observing changes in protein function It is also used to create or delete restriction sites to be used in DNA cloning. Also, in order to know what factors combine to function in the promotor or enhancer studies, we can change the base sequence of the binding site to prevent binding, and then measure the activity of the promotor or enhancer There is. The Muta-Direct™ Site-Directed Mutagenesis Kit provides the most optimized Protocol (see Technical Data) to facilitate mutation even for non-specialist non-specialists, as well as enabling low-cost experiments.
|Muta-Direct™ Enzyme (2.5 U/ml)||15 ul x 1 tube|
|Muta-Direct™ Reaction Buffer (10x)||100 ul x 1 tube|
|dNTP Mixture||30 ulx 1 tube|
|Mutazyme™ Enzyme (10 U/ml)||15 ul x 1 tube|
|pUC18 Control Plasmid (10 ng/ml)||10 ul x 1 tube|
|Control Primer Mix (20 pmole/ml)||10 ul x 1 tube|
1. Generation of primers for mutation of the desired site (see primer design of the
2. PCR amplification (15-18 cycles) using Muta-Direct™ enzyme obtain mutation clone by mutazyme™ treatment
3. Transfection in E. coli Spreading on an LB plate containing a specific antibiotic (the theoretically formed clone is a 100% mutagenized cl
4. Final confirmation of mutation through sequencing
1. Primer design : Primer size is within 25 ~ 45 mer and 30 ~ 35 mer is recommended. The
important thing is that the nucleotide has designed to be located at the center of the primer and designed to be 30-mer, so that the
following Tm-A is above 78 ℃ (GC% is at least 40%) If the Tm value does not exceed 78 ℃, increase or decrease the primer size.
1) Design forward and reverse primers with mutually complementary sequences, where the desired mutation sites are located.
2) Design a 30-mer to check if the Tm value is 78 ℃ or more. If less, increase the primer size to 78 ℃ or more.
3) Do not use desalting grade for primer, but use minimum FPLC or OPC refined purity.
2. Tm calculation formula: 0.41 (GC%) - 675 / L + 81.5 L: Number of oligomers of the primer GC%: Primer% of GC base
3. Example of primer design: The following is an example of primer design, where GCG → ACG.
5 '- CCTCCTTCAGTATGTAGGCGACTTACTTATTGCGG-3' Design the forward and reverse primers for 30mer each, with "A (or T)" at the center for mutation. forward Primer: 5'-CCTTCAGTATGTAGACGACTTACTTATTGC-3 ' reverse primer: 5'- GCAATAAGTAAGTCGTCTACATACTGAAGG-3 ' The above primer GC% = 40 and L = 30, and it is calculated by the above Tm calculation formula to be Tm = 0.41 * 40 - 675/30 + 81.5 = 75.5 ° C, In such cases, increase or decrease the primer size Forward Primer: 5'-CCTCCTTCAGTATGTAGACGACTTACTTATTGCGG-3 ' reverse primer: 5'- CCGCAATAAGTAAGTCGTCTACATACTGAAGGAGG-3 'In the above example, GC% = 45.7 and L = 35 when the 5-mer is increased, Tm = 0.41 * 45.7 - 675/35 + 81.5 = 80.952 ° C, indicating that the Tm is 78 ° C or higher, which is a suitable primer.