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Muta-Direct™ Site Directed Mutagenesis Kit

Cat.No	Capacity	Inquire
15071	15 rxn.

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Description
TroubleShooting Guide
Related products
Protocol
MSDS

PRODUCT INFORMATION

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

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

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

The example below is for mutagenesis with a mutation of GCG → ACG.

TroubleShooting Guide

QLow transformation efficiency or low colony number?

A- Insufficient amount of DNA template used in the reaction.
→ Visualize the DNA template on a gel to verify the quantity and quality.

- Too much mineral oil pipetted with the Mutazyme™ Enzyme-treated DNA while transferring to the transformation reaction.
→ Using the smallest pipet tips available, insert the pipet tip completely below the mineral layer overlay and clear the pipet tip while submerged beneath the mineral oil overlay before collecting the sample.

- Insufficient amount of mutant DNA synthesized in the reaction.
→ Increase the amount of the Mutazyme™ Enzyme treated DNA used in the transformation reaction to 4μl.

Q Low mutagenesis efficiency or low colony number with the control reaction?

A - Little or no linear amplification products.
→ Following temperature cycling, resolve a sample of the control reaction by electrophoresis on an agarosegel; if no product is observed at 4.5kb, adjust the cycling parameters for the control reaction.

- Insufficient amounts of X-gal and IPTG on the agar plates.
→ Prepare the LB–ampicillin agar plates for the transformed control cells by pipetting 20μl of 10% (w/v) X-gal (prepared in DMF) and 20μl of 100mM IPTG (prepared in filter-sterilized dH2O) into a 100-μl pool of NZY+ broth and then spreading the mixture across the plate.

- Competent cells stored at improper temperature.
→ Store the competent cells immediately at the bottom of a –80°C freezer.

- Differences in thermal cyclers may contribute to variations in ramping efficiencies.
→ Adjust the cycling parameters for the control reaction and repeat the protocol for the sample reactions. For the best visualization of the blue (β-gal+) phenotype, the control plates must be incubated for at least 16 hours at 37°C.

- Subjecting the dNTP mix to multiple freeze-thaw cycles.
→ Thaw the dNTP mix once, prepare single-use aliquots, and store the aliquots at –20°C. Do not subject the dNTP mix to multiple freeze-thaw cycles.

Q Low mutagenesis efficiency with the sample reaction(s)?

A - Differences in thermal cyclers may contribute to variations in ramping efficiencies.
→ Adjust the cycling parameters for the sample reaction.

- Improper mixing of reagents.
→ Add the Mutazyme™ Enzyme restriction enzyme below the mineral oil overlay in the digestion step and ensure proper mixing of all components in the reaction especially the Mutazyme™ Enzyme.

- The amplification reaction contains too many DNA template.
→ The Mutazyme™ Enzyme must be able to completely digest the parental template in the time allotted for the digestion; repeat the digestion if necessary.

Subjecting the dNTP mix to multiple freeze-thaw cycles.
→ Thaw the dNTP mix once, prepare single-use aliquots, and store the aliquots at–20°C. Do not subject the dNTP mix to multiple freeze-thaw cycles.

The parental plasmid DNA was notmethylated.
→ Check the Host E. coli Strain. If the host strain has dcm or dam gene, the parental plasmid DNA will not be methylated. Therefore the Mutazyme can not be activated to digest parental DNA.

Q False positives?

A - Quality of the primers is poor.
→ Radiolabel the primers and check for degradation on an acrylamide gel or re-synthesize the primers.

- False priming.
→ Increase the stringency of the reaction by increasing the annealing temperature to within 5°C of the melting temperature of the mutation primers.

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