KM101-Fast Site-Directed Mutagenesis Kit

Fast Site-Directed Mutagenesis Kit
——For efficient introduction of single or multiple mutations on target gene

Fast Site-Directed Mutagenesis Kit
Cat. No. 4992901

Storage
FDM competent cells: Please store the FDM competent cells at -90~-65°C
right after you receive it. Cells would be stable for 6 months at -90~-65°C.
All other components should be stored at -30~-15°C, at which they are
stable for one year.

Introduction
In vitrosite-directed mutagenesis is a crucial technique for the modification
and optimization of target gene, studying regulatory site of promoter and
the complicated relationship between protein’s structure and function.
The Fast Site-Directed Mutagenesis Kit allows site-specific mutation at
target gene, including single and multiple mutations, insertion or deletion
mutations. The Fast Site-Directed Mutagenesis Kit can promise a 90%
or even higher mutation rate for introducing a single mutation to target
gene. Different from previous low efficient mutagenesis kit, this Fast SiteDirected Mutagenesis Kit does not require multi-round PCR or sub-clones
which cost lots of time and labor; only four steps are required to construct
mutants (Figure 1).

Figure 1. Overview of the Fast Site-Directed Mutagenesis system
FastAlteration DNA Polymerase in this kit is a DNA polymerase with high
fidelity, high speed and high sensitivity. It could amplify up to 10 kb of
plasmid at 15-30 sec/kb. FDM competent cells could digest methylated
plasmid within the cell, it means that they could degrade those plasmids
which are failed to be degraded by DpnI and then ensure a higher positive
rate. Meantime, this kit also contains control plasmid and primers to help
customers to locate experimental problems.

Features

1. Simple and fast:Only four steps are required to obtain mutants. Unlike
   traditional strand displacement amplification (SDA), this kit does not
   require multi-round PCR or the construction of sub-clones.
2. High-efficiency primers:Part-overlapping primers were designed to
   generate more desired mutated plasmids by PCR amplification.
3. Wide applications:Both single and multiple mutations can be applied
   by using this kit, and the mutation sites can be up to five within a single
   plasmid.
4. High suitability:Size of the target plasmid can be up to 10 kb, which
   means almost all the common used plasmids suit to this kit.
5. High mutation rate:Methylated plasmid can be digested both in vivo
   and in vitroby using this kit, ensuring higher mutation rate. And for
   single mutation, the mutation rate is higher than 90%.

Principles of primer design

1. Both of the mutagenic primers must have the desired mutation sites on
   them, and the primers’ sequences should be complementary with the
   target plasmid except for the mutation site.
2. If there is only one mutation site in a primer, it should be designed as
   what is showed in Figure 2-A. This kind of primer should contain two
   parts: 5’ overlapping area and 3’ extension area. Primers should be
   around 30 bases in length which include 15-20 bases of 5’ overlapping
   area and at least 10 bases of 3’ extension area. The mutation site should
   locate at the downstream of forward primer’s overlapping area and at
   the 5’ end of reverse primer.
3. If there are 2-5 mutation sites in a primer, it should be designed as what
   is showed in Figure 2-B. The sequences of this primer pair are completely
   complementary and contain two parts: mutation area and non-mutation
   area. The length of primer should be around 40 bases, within which the
   mutation area should be 15-20 bases and the non-mutation area should
   be at least 10 bases. 2-5 mutation sites could be applied depending on
   the experimental purpose.
4. Primers must be purified either by high performance liquid
   chromatography (HPLC) or by polyacrylamide gel electrophoresis (PAGE).
5. The following formula is commonly used for estimating the Tm of
   primers:

Important Notes:

1. When multiple mutations are applied on a single primer, the more
   mutation sites it contains, the lower mutation rate occurs. Previous
   data suggested that when five mutation sites were designed on a single
   primer, the mutation rate were around 50%. We suggest increasing the
   clones number to verify.
2. This kit allows multiple mutations in multiple primers, which gives a
   wider range for mutation study in a certain gene. The upper limit of
   mutation sites is still five.
3. Control plasmid and primers are strongly recommended to be used for
   the troubleshooting reason.

Applications
This kit could be used to optimize the target gene or vector, analyze the
sites on promoter which interact with regulatory proteins, and study the
relationship between the structure and function of proteins.

KM101-Fast Site-Directed Mutagenesis Kit

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This product is for scientific research use only. Do not use in medicine, clinical treatment, food or cosmetics.

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