Muta-Direct™ Site Directed Mutagenesis Kit

Description

Description

The Muta-Direct™ Site-Directed Mutagenesis Kit can be used for creating a mutation at a defined site in a plasmid. It has convenient and simple three steps for all experimental procedures.

The Muta-Direct™ Site-Directed Mutagenesis Kit is used to make point mutations, substitute amino acids, and delete or insert nucleotide sequence(s). Its characteristics enable the kit to be applicable to protein engineering including the improvement of protein function or protein productivity as well as analysis of genefunction.

The creation of a mutation is possibly to complete through just simple three-steps including performance of PCR using the prepared mutagenic primers and use of the Muta-direct™ enzyme which has a very low error rate with proof-reading function; digestion of non-mutated parental DNA template (contained with methylated and hemimethylated DNA sequence) by treatment with Mutazyme™ Enzyme; and transformation of the mutated plasmid (Fig. 1). It can be checked whether mutagenesis is completed or not by sequencing of mutated plasmid if necessary.

The Muta-Direct™ Site-Directed Mutagenesis Kit provides a simplified user-friendly protocol for the convenient use for those who are unfamiliar with or new to the Kit.

Characteristics

• Easy to use : No special skills are required

• Simple : Three steps with two days ​

• High mutation efficiency : The success rate is 99%

Application

• Codon switch

• Re-mutation to wild type of plasmid

• Functional analysis of a gene or protein

• Protein engineering

Kit Contents

No.	Kit Contents	Unit
1	Muta-Direct™ Enzyme (2.5U/μl)	15μl
2	Muta-Direct™ Reaction Buffer(10X)	100μl
3	dNTP Mixture	30μl X 1 bottle
4	Mutazyme™ Enzyme (10U/μl)	15μl
5	pUC18 Control Plasmid (10ng/μl)³	10μl
6	Control Primer Mix (20 pmole/μl)³	10 μl
7	Manual	1 ea

Analysis Workflow

Important Points Before Starting

1. Muta-Direct™ Control Reaction

The pUC18 Control Plasmid and Control Primer Mix contained in the Muta-Direct™ Site-Directed Mutagenesis Kit are provided to check whether the mutagenesis experiment is performed completely. The pUC18 Control Plasmid and Control Primer Mix should be used in the Muta-Direct™ Control Reaction.

Through the Muta-Direct™ Control Reaction, a translational termination codon is introduced into the lacZ gene contained in pUC18 Control Plasmid. The change from serine (TCG) to translational termination codon (TAA) can block the expression of protein product of the lacZ gene. Only white colonies are formed after transformation when the experiment completes properly.

2. Primer Design

At first, design of the mutagenic primers is required for the use of the Muta-Direct™ Site- Directed Mutagenesis Kit.

It is generally accepted that the length of mutagenic primers is 25~45 bp. We recommend the use of the mutagenic primer which is 30-35 bp in length. It is important that the nucleotide desired to be mutated should be settled in the middle of the mutagenic primers.

Design a primer of 30 bp in length and then need to estimate a melting temperature(Tm) with Tm formula. Tm of the mutagenic primers should be greater than or equal to 78°C (At least more than 40% of GC ratio). If the Tm is under 78°C, the change of the primer length is necessary.

Note : Check points below for the design of primer.

1) Design forward and reverse primers which are 30 bp each in length. In this step, locate the nucleotide desired to be mutated in the middle of the mutagenic primers.

2) Estimate the Tm of the mutagenic primers. If the Tm is under 78℃, adjust the length of primers for 78°C (At least more than 40% of GCratio).

3) Avoid using desalting-grade primers. It is recommended to use HPLC or PAGE grade of primers. Most companies commonly provide HPLC grade primer but customers are required to check this point.

The following formula is commonly used for estimating the Tm of mutagenic primers.

Mutagenesis example

Available pack options: 15 rxn.. Manufacturer reference: 15071.