Traditional subcloning
  • OVERVIEW

Traditional subcloning

Seattle Genova provides fast and efficient molecular cloning services to our customers. Our services include subcloning a gene of interest into a vector of choice, site-directed mutagenesis, small insertions, and deletions. Molecular biologists at Seattle Genova are experts in molecular cloning and our work aim to reduce the time, effort and money spent by researchers. Simply provide us your project details and we will deliver ready-to-use DNA vectors generated according to your exact specifications.

There are many different methods, technologies, and protocols are available for performing the actual cloning reaction. The following guide will highlight several of the most popular cloning methods used to create recombinant DNA.

1.1. Restriction Enzyme Cloning

· In restriction cloning, scientists utilize specific restriction enzymes to cut dsDNA of interest into fragments containing precise 5' or 3' single-strand overhangs (sticky ends), or no overhang (blunt ends).

· Two pieces of DNA that have complementary overhangs, or which are both blunt-ended, can then be fused together during a ligation reaction with T4 DNA ligase.

· Can easily move (subclone) any piece of DNA that already has restriction sites on either side of it into any plasmid that has the same sites in the same orientation within its multiple cloning site.


1.2. Gateway® Recombination Cloning

· Your DNA fragment must first be amplified with specific Gateway attB1 and attB2 sites attached to the 5’ and 3’ ends of DNA sequence.

· This fragment can then be cloned into a gateway donor plasmid which contains compatible attP sites via a proprietary BP clonase (creating an entry clone). The entry clone now has recombined attL sites flanking your DNA fragment of interest.

· The entry clone can be rapidly shuttled into any compatible Gateway® Destination vector, which contain attR sites via LR clonase enzymes.


1.3. TOPO® Cloning

· TOPO® cloning utilizes the Taq polymerase which naturally leaves a single adenosine (A) overhang on the 3' end of PCR products.

· The complementary thymine (T) comes from a pre-cut, linear, cloning ready TOPO® vectors that has a DNA topoisomerase I fused to the 3’ end. The topoisomerase acts as a ligase that joins the A and T compatible ends together.

· TOPO® cloning thus does not need restriction enzymes or an exogenous ligase providing an incredibly quick and easy way to clone a fresh PCR product into a plasmid.


1.4. Gibson Assembly (Isothermal Assembly Reaction)

· takes advantage of the properties of 3 common molecular biology enzymes: 5' exonuclease, polymerase and ligase.

· 5' exonuclease digests the 5' end of dsDNA fragments to generate 3' single-stranded overhangs. DNA polymerases synthesize DNA molecules using the 4 nucleotides and lastly DNA ligase fuses DNA strands together.


1.5. Golden Gate Cloning

· take advantage of the unique properties of type IIS restriction endonucleases. These endonucleases cut dsDNA at a specified distance away from the recognition sequence. Cutting distal to their recognition site allows for the creation of custom overhangs, which is not possible with traditional restriction enzyme cloning. Scientists have utilized this approach to create compatible custom overhangs that can then be efficiently assembled together.


1.6. Ligation Independent Cloning

· In LIC, the T4 DNA polymerase’s exonuclease activity creates “chewed-back” overhangs of 10-12 base pairs on the 5' end of both the vector and insert. These overhangs can easily anneal creating a circular product with four nicks that are repaired by the bacteria after transformation. LIC does not require site-specific recombination or a ligation step, making it an easy, cheap and rapid cloning method.


1.7. Yeast-mediated Cloning and Oligonucleotide Stitching

· can efficiently fuse two (or more) fragments of dsDNA that have 30 or more bases of overlapping homology. One major advantage is that much larger final products can be generated (up to 100kb) compared to other cloning methods that utilize bacteria where it becomes progressively more difficult to clone plasmids larger than 10kb.


Delivery Specifications

All of our Custom Cloning services guarantee 100% sequence fidelity and deliver everything you need to put your new constructs to work right away. Our delivery package includes the following:

· 10 μg of constructed plasmid

· Project report including sequence design and construction methods

· Sequencing data

· Vector map

Traditional subcloning

Seattle Genova provides fast and efficient molecular cloning services to our customers. Our services include subcloning a gene of interest into a vector of choice, site-directed mutagenesis, small insertions, and deletions. Molecular biologists at Seattle Genova are experts in molecular cloning and our work aim to reduce the time, effort and money spent by researchers. Simply provide us your project details and we will deliver ready-to-use DNA vectors generated according to your exact specifications.

There are many different methods, technologies, and protocols are available for performing the actual cloning reaction. The following guide will highlight several of the most popular cloning methods used to create recombinant DNA.

1.1. Restriction Enzyme Cloning

· In restriction cloning, scientists utilize specific restriction enzymes to cut dsDNA of interest into fragments containing precise 5' or 3' single-strand overhangs (sticky ends), or no overhang (blunt ends).

· Two pieces of DNA that have complementary overhangs, or which are both blunt-ended, can then be fused together during a ligation reaction with T4 DNA ligase.

· Can easily move (subclone) any piece of DNA that already has restriction sites on either side of it into any plasmid that has the same sites in the same orientation within its multiple cloning site.


1.2. Gateway® Recombination Cloning

· Your DNA fragment must first be amplified with specific Gateway attB1 and attB2 sites attached to the 5’ and 3’ ends of DNA sequence.

· This fragment can then be cloned into a gateway donor plasmid which contains compatible attP sites via a proprietary BP clonase (creating an entry clone). The entry clone now has recombined attL sites flanking your DNA fragment of interest.

· The entry clone can be rapidly shuttled into any compatible Gateway® Destination vector, which contain attR sites via LR clonase enzymes.


1.3. TOPO® Cloning

· TOPO® cloning utilizes the Taq polymerase which naturally leaves a single adenosine (A) overhang on the 3' end of PCR products.

· The complementary thymine (T) comes from a pre-cut, linear, cloning ready TOPO® vectors that has a DNA topoisomerase I fused to the 3’ end. The topoisomerase acts as a ligase that joins the A and T compatible ends together.

· TOPO® cloning thus does not need restriction enzymes or an exogenous ligase providing an incredibly quick and easy way to clone a fresh PCR product into a plasmid.


1.4. Gibson Assembly (Isothermal Assembly Reaction)

· takes advantage of the properties of 3 common molecular biology enzymes: 5' exonuclease, polymerase and ligase.

· 5' exonuclease digests the 5' end of dsDNA fragments to generate 3' single-stranded overhangs. DNA polymerases synthesize DNA molecules using the 4 nucleotides and lastly DNA ligase fuses DNA strands together.


1.5. Golden Gate Cloning

· take advantage of the unique properties of type IIS restriction endonucleases. These endonucleases cut dsDNA at a specified distance away from the recognition sequence. Cutting distal to their recognition site allows for the creation of custom overhangs, which is not possible with traditional restriction enzyme cloning. Scientists have utilized this approach to create compatible custom overhangs that can then be efficiently assembled together.


1.6. Ligation Independent Cloning

· In LIC, the T4 DNA polymerase’s exonuclease activity creates “chewed-back” overhangs of 10-12 base pairs on the 5' end of both the vector and insert. These overhangs can easily anneal creating a circular product with four nicks that are repaired by the bacteria after transformation. LIC does not require site-specific recombination or a ligation step, making it an easy, cheap and rapid cloning method.


1.7. Yeast-mediated Cloning and Oligonucleotide Stitching

· can efficiently fuse two (or more) fragments of dsDNA that have 30 or more bases of overlapping homology. One major advantage is that much larger final products can be generated (up to 100kb) compared to other cloning methods that utilize bacteria where it becomes progressively more difficult to clone plasmids larger than 10kb.


Delivery Specifications

All of our Custom Cloning services guarantee 100% sequence fidelity and deliver everything you need to put your new constructs to work right away. Our delivery package includes the following:

· 10 μg of constructed plasmid

· Project report including sequence design and construction methods

· Sequencing data

· Vector map

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