30 rxn

30.00

Description

Two crucial procedures in cloning are the ligation of the foreign DNAs to the vector DNA and the transformation of bacteria using those ligated DNA constructs (the recombinant molecules). Ligation is accomplished using the enzyme DNA ligase (usually from the bacteriophage T4). It requires ATP and magnesium ions to catalyze the reaction of a 3'-OH and a 5'-P on double-stranded DNA to form a phosphodiester bond. The DNA ends can be cohesive ends, such as those formed between molecules that have been digested with the same restriction endonuclease, or they can be blunt ends. Ligation between cohesive-ended molecules is much more efficient than ligation between blunt-ended molecules. Because of this, when ligatiog blunt-ended molecules, the DNA concentration must be higher than when ligating cohesive-ended molecules. The rates of blunt-end and cohesive-end ligation of DNA by T4 DNA ligase are increased by orders of magnitude in the presence of Polyethylene glycol (PEG) [Pheiffer, B. H., Zimmerman, S. B., Nucleic Acids Res., 11, 7853-7871, 1983]. The Rapid T4 DNA Ligase is based on this PEG-assisted rapid ligation of DNA and enables sticky- and blunt-ended DNA ligation in only 10 min. 5×Rapid Ligation Buffer (RLB) supplied in this kit is specially designed for the efficient rapid ligation of DNA. For an unknown reason, while ligation efficiency appears to increase for high PEG concentration, the transformation efficiency for molecules ligated in this condition decrease. Therefore, appropriate PEG concentration should be selected. At the PEG concentration should be selected. At the PEG concentration utilized in the Rapid T4 DNA Ligase, ligation and transformation are  both increased significantly.

Storage
All components should be stored at -20℃. Do not substitute the reaction buffer supplied in this kit with other one.

Contents

For research use only. The Rapid DNA Ligaton Kit contains sufficient reagents to perform approximately 30×20 μl DNA ligaton reactions.

Characteristics

Sufficient ligation efficiency can be archived at room temperature (20-25℃) for 10-30 min.

Storage Buffer

Quality Control

T4 DNA ligase supplied in this kit is free of detectable exo- or endonuclease activities. Each lot is functionally tested in the ligation reaction. Additionally, each lot is analyzed by SDS polyacrylamide gel electrophoresis for the presence of detectable contaminating proteins (less than 5%).

Exonuclease assay:

After incubation of a 50 μl reaction containing T4 DNA ligase with 1 μg of sonicated [3H]-labeled E.coli DNA (105 cpm/μg) for 4 hours at 37℃. The DNA is precipitated with trichloroacetic acid and the radioactivity of the supernatant is evaluated. Exonuclease activity is expressed as a percent of total DNA radioactivity released into the acid soluble fraction. The exonuclease activity was determined to < 0.1% radioactivity. The limit of detectability of this assay is approximately 0.05%.

Endonuclease assay:

Incubation of a 50 μl reaction containing T4 DNA ligase with 1 μg of supercoiled plasmid DNA for 4 hours at 37℃ resulted in < 0.1% conversion to nicked or linear plasmid DNA as determined by agarose gel electrophoresis.

Protocol

     A. Cloning of DNA fragments into plasmid vectors

We recommend using a 1:1 or 1:3 molar ratio of vector: insert DNA. These ratios will vary with the types of vectors, for example, cDNA and genomic DNA cloning vectors. To calculate the required amount of insert DNA, the following formula can be used.

[Example]

How much 0.5 kbp insert DNA should be added to a ligation in which 100 ng of 6 kbp vector will be used? The desired vector: insert ratio will be 1:3.  

1. Prepare the following reaction in a sterile microcentrifuge tube:

2. Incubate the reaction at room temperature for 10 min.

3. Competent E. coli was transformed with the ligation mixture.

     (Competent cell efficiency: > 1×10⁷)

     B. Recircularization of linear DNA

The amount of DNA is important in recircularization. For the enhanced recircularization, lower DNA concentration is helpful compared to the cloning of DNA fragment into plasmid vectors because low DNA concentration makes more intramolecular ligation (recircularization) and suppress intermolecular ligation. Intramolecular ligation is Concentration-independent.

[Example]

1. Prepare the following reaction in a sterile microcentrifuge tube:  

2. Incubate the reaction at room temperature for 10 min.

3. Competent E. coli was transformed with the ligation mixture.

     (Competent cell efficiency: > 1×10⁷)

     C. Adaptor ligation

Adaptor ligation conditions are basically the same as for cloning of DNA fragments into plasmid vectors (8 bases or longer). However, if the adaptor is shorter than 8 bases or if GC-contents are low in adaptors, the ligation reaction should be carried out at 16℃ for 30minutes to 2 hours. We recommended vector/linker molar ratio is: Dephosphorylated vector: Phosphorylated linker > 1:100

[Example]

1. Prepare the following reaction in a sterile microcentrifuge tube:

2. Incubate the reaction at 16℃ for 1 hour.

3. Competent E. coli was transformed with the ligation mixture.

     (Competent cell efficiency: >1×10⁷)

or

In the case of adaptor ligation to termini, inactivate T4 DNA ligase by heating at 70℃ for 10 min and appropriate purification is performed for user’s purpose.

There are many problems with ligation reaction itself and with the competent cells, the selection medium, the restriction endonuclease digestion of the vector, and the phosphatase treatment of the vector. Because transformation of some competent cells is inhibited by components of the ligation reaction, the reaction should be diluted fivefold before being used for transformation. Some possible causes of unsuccessful ligation are listed next along with suggested solutions.

15 rxn

119.00

Description

The Site-Directed Mutagenesis Kit can induce mutagenesis at the specific point of sequence that cloned on plasmid DNA. It guarantees 100% of efficiency in theory. Also it is very convenient and simple because it takes just two steps for all experimental procedures. The Site-Directed Mutagenesis Kit does not necessary using M13 vector and methylation step. Indeed, the Kit can induce mutation of nucleotide, re-mutation to wild type, mutation of codon and insertion even deletion. As the Kit has these characteristics, it is applicable to analysis for genomic/proteomic function. Also as inducing mutagenesis of specific gene, it can be used for protein engineering like protein development or improving productivity.

When you use this the Kit, you can have mutated clone as doing simple steps. (Design primer with own protocol, use the Enzyme for 15~18 cycles of PCR. Proceed transformation step after the Mutazyme treatment for mutated clone selection) In this theory, clones on LB agar plate are mutated around 100% and after sequencing, you can proceed to the next step.

Characteristics

Without special skill, easy to use.

Can induce mutagenesis within 2~3 days.

Use only two enzymes: Muta-direct™ Enzyme and Mutazyme™ Enzyme.

Can use for various experiment: Point mutation, Deletion, Insertion and etc.

100% of mutation efficiency.

Reasonable price.

Technical assist by research agents.

Storage and Stability

All component should be stored at -20℃. The reaction buffer and dNTP mixture have been optimized for the Muta-direct™ protocols.

Kit Contents

For research use. The Muta-direct™ kit contains sufficient reagents to perform approximately15×50μl mutagenesis reactions. The kits contain enough control template and primer mix for 5 control reac-tion, and enough reagents for 15 reactions total (control and experimental reactions combined).

Muta-direct™ Control Reaction

Control plasmid, contained in Muta-direct™, is pUC18 that informs us whether the experiment success or not. pUC18 plasmid has lacZ gene, so we can confirm the result as induce termination codon at lacZ gene by using Control Primer Mix (provided). In case of success, there must be all white colonies on LB plate. As change from serine (TCG) to stop codon (TAG) in pUC18, lacZ gene can be blocked.

If user handles the mutation procedure for the first time, he can know about result as proceed of the control reaction step.

Primer Design

At first, it is required to design a primer. It is no matter who has experiences about designing. Just have a check the points below when you want design your primer.

Normally, primer size is 25~45mer and we recommend 30~35 mer length. The important thing is that the target nucleotide on the center of primer.

Design as 30mer and next, you have to calculate the Tm value, more than 78℃ or not. (At least more than 40% of GC ratio).

If the Tm value is under 78℃, it is necessary to change the primer length.

① Design two strands, forward and reverse primers. In this step, locate the target nucleotide on the center of primer.

② Calculate the Tm value to know whether over than 78℃ or not. If the value is under 78℃, adjust the length of primer for 78℃ (Minimum GC ratio is 40%).

③ Avoid desalting grade, Must use over than minimum FPLC or OPC grade. Normally, the most of companies use OPC but it depends on the company. So customer must check this point.

Tm formula: Tm = 0.41(% of GC) – 675/L + 81.5

L: Number of oligomer in primer, % of GC: GC % of primer

Primer Design Example

Next, showing primer design.  Case of GCG → ACG.

       5' CCTCCTTCAGTATGTAGGCGACTTACTTATTGCGG-3'

① First step, locate A (or T) to center which you want to mutate and then design 30mer for forward and reverse each.

Primer #1: 5'-CCTTCAGTATGTAGACGACTTACTTATTGC-3'

Primer #2: 5'-GCAATAAGTAAGTCGTCTACATACTGAAGG-3'

② This primer contain 40% of GC and L value is 30, using these data to Tm formula, the result is 75.5℃(Tm=0.41×40-675/30+81.5). So we can find that the Tm value is under 78℃. This is not an appropriate primer.

③ In this case, it is necessary to adjust the length of primer.

Primer #1: 5'-CCTCCTTCAGTATGTAGACGACTTACTTATTGCGG-3'

Primer #2: 5'-CCGCAATAAGTAAGTCGTCTACATACTGAAGGAGG-3'

5 mers are added to original primers (italic, under lined). In this case, the primers contain 45.7% of GC and L value is 35, using these data to Tm formula, the result is 80.952℃(Tm=0.41×45.7-675/35+81.5). Now you can use this primer.

Muta-direct™ PROTOCOL

[A] Induction of Mutagenesis (PCR Reaction)

In this step, you can induce mutagenesis at target nucleotide. As using synthesized primer, proceed PCR reaction with Muta-direct™ Enzyme.

1. Design each primer for Site direct mutation.

[Note] Refer to Primer design guide.

2. Prepare plasmid DNA as a template.

[Note] Use dam⁺ bacteria (ex. DH5 strain) as host. (Almost dam⁺ bacteria when except JM110 and SCS110 strain). Also, in case of end⁺ strain, sometimes it can be happen the number of colony is low. But this is not affected to mutation efficiency. We recommend to use DNA-spin™ and DNA-midi™ Plasmid DNA extraction kit when you extract plasmid DNA.

3. [Option] Control reaction (50μl reaction volume)

10×reaction buffer                                                           5μl

pUC18 control plasmid (10ng/μl, total 20ng)                       2μl

Control primer mix (20pmol/μl)                                          2μl

dNTP mixture (each 2.5mM)                                             2μl

dH₂O                                                                             38μl

Muta-direct™ Enzyme                                                     1μl

4. Sample reaction (50μl reaction volume)

10×reaction buffer                                                            5μl

Sample plasmid (10ng/μl, total 20ng)                                  2μl

Sample primer (F) (10pmol/μl)                                            1μl

dNTP mixture (each 2.5mM)                                               2μl

dH₂O                                                                               38μl

Muta-direct™ Enzyme                                                       1μl

5. PCR condition

[Note] Follow the PCR condition described below and final extension step can be omitted.

6. After PCR, put it in the ice for 5 minutes then, store at RT.

(Avoid frequent freeze-thawing).

[Note] In the PCR condition described above, control the number of PCR cycle.

Note that there is very low mutagenesis efficiency in case more than 4 nucleotides are mutated or Avoid more than 4 nucleotides mutagenesis. In this case, the efficiency can be very low.

[B] Selection of mutated plasmid

In this step, you can select mutated plasmid DNA by digestion of the methylated plasmid with Mutazyme™ Enzyme after PCR reaction.

1.  Prepare the product from above PCR reaction.

2.  Incubate the sample at 37℃ for 1hour with 1μl (10U/μl) of Mutazyme™ Enzyme.

[Note] In case of using much amount of plasmid DNA, sometimes Mutazyme™ Enzyme couldn’t reaction with sample. So we suggest you to follow the procedure correctly for good mutation efficiency. If mutation efficiency is low, take a long time for reaction or add more amount of Mutazyme™ Enzyme.

[C] Transformation

This step recovers the nick on the plasmid DNA after reaction. When you transform into E.coli, use dam+ strain competent cell like DH5a.

1.  Put the 10μl sample into 50μl competent cell vial and than keep it in the ice for 30minutes.

2.  Follow general steps with an appropriate transformation method.

SEQUENCING ANALYSIS
White colonies on LB plate resulted by Muta-direct™ protocol is supposed to be occurred 100% of mutation.

To confirm this result, sequencing analysis is recommended with white colonies.

Example of mutagenesis inducing.  GGC→GAC

[Sequencing Analysis]

Sequencing result of mutated plasmid