Subcloning by restriction digestion is a commonly used laboratory technique. Our technique can be used to easily move any DNA fragment from one vector to another, as long as it has been restricted by restriction sites that are also present in the same orientation on the target vector.

Design

Creative Enzymes has access to many DNA analysis tools that allow you to identify which restriction sites are present in a given sequence. When choosing a restriction endonuclease, you will need to select one that has

• Side cut your blade, but not inside your blade
• Cuts at the desired location in your recipient plasmid, but not elsewhere in the plasmid
• Conform to the frame of the tag or fusion protein in the recipient plasmid

Workflow

• Digesting your DNA

Set up restriction digests for your donor and recipient plasmids. Because some DNA is lost during the gel purification step, it is important to digest a large amount of starting material. We recommend 1.5-2 μg of donor plasmid and 1 μg of acceptor plasmid. It is also important to cut as much of the acceptor plasmid as possible with both enzymes, so it is important to digest for at least 4 hours and up to overnight.

• Isolation of insert fragments and vectors by gel purification

Run the digested DNA on an agarose gel and perform gel purification to isolate the DNA.

• Ligate your insert into your vector

Perform DNA ligation to fuse the insert to the recipient plasmid. We recommend using approximately 100ng of total DNA in a standard ligation reaction. ideally, you want the recipient plasmid insert to be at a ratio of approximately 1:3.

• Transformation

Transform your ligation reaction into the bacterial strain of your choice. For most standard clones, you can transform 1-2 μl of the ligation reaction into receptor cells

• Isolation of the completed plasmid

Finally, you need to pick individual bacterial colonies and check if they are successfully ligated. Pick 3-10 colonies based on the number of background colonies on the control plate (the more background, the more colonies you need to pick) and incubate overnight for DNA purification.

Service Process

Our Features

• We have accumulated many years of essence in the field of plasmid cloning service by restriction enzymes service, helping customers to speed up project development and improve the overall success rate of the project.
• At Creative Enzymes, simply let us know your specific project needs. We will suggest the best strategy for you.

Deliverables

• Related experimental results raw data
• Experimental report
• Data analysis
• Image and result analysis
• Results Analysis

Why Choose Us?

Creative Enzymes is a company that provides professional and comprehensive plasmid cloning service by restriction enzymes service. We have years of experience to meet your specific project needs in using enzyme research to add value to your research projects. Creative Enzymes can provide you with personalized solutions to help you thrive every step of the way around your interest in your workflow. If you would like to know more about this service, please feel free to contact us.