Cloning Basics Support - Getting Started

An ori, or origin of replication, is the sequence where replication is initiated. The ori determines the vector copy number. For example, a pUC ori produces a high copy number, whereas a pBR322 ori produces a low copy number. An ori is necessary for replication in bacteria.

The pCR™II vector is a dual promoter vector: it contains a T7 promoter at the 5' end of the multiple cloning site and an Sp6 promoter at the 3' end. The pCR™2.1 vector only contains the T7 promoter (the Sp6 promoter was removed). Having a dual promoter vector is only of advantage if you want to do in vitro transcription studies with your insert. In terms of cloning efficiency, there is no difference between the pCR™2.1 and the pCR™II vectors. Both the pCR™2.1 and the pCR™II vectors contain sequencing primer sites (M13 primer sites) to sequence your insert in both directions.

TA Cloning

This cloning method was designed for use with pure Taq polymerases (native, recombinant, hot start); however, High Fidelity or Taq blends generally work well with TA cloning. A 10:1 or 15:1 ratio of Taq to proofreader polymerase will still generate enough 3’ A overhangs for TA cloning.

Recommended polymerases include Platinum™ Taq, Accuprime™ Taq, Platinum™ or Accuprime™ Taq High Fidelity, AmpliTaq™, AmpliTaq Gold™, or AmpliTaq Gold™ 360.

Blunt Cloning

Use a proofreading enzyme such as Platinum™ SuperFi™ DNA Polymerase..

Directional TOPO Cloning

Platinum™ SuperFi™ DNA Polymerase works well.

T4 DNA polymerase and Klenow fragment of E. coli DNA polymerase can both convert overhangs to blunt molecules. The 3’ – 5’ exonuclease activity of the T4 DNA polymerase is much more efficient than Klenow.

CIP/CIAP or BAP can dephosphorylate the 5’ ends of DNA/RNA. T4 Polynucleotide Kinase can add back phosphate groups.

Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. The Shine-Dalgarno sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA.

Eukaryotic (and specifically mammalian) mRNA contains sequence information that is important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:

• Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
• Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
• Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
• Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
• Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.

Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:

• Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
• Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.

Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. The Shine-Dalgarno sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information that is important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:

• Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
• Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
• Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
• Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
• Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.

Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:

• Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
• Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.

ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/ribosome binding site (RBS) or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.

An IRES is an internal ribosome entry site, which allows for end-independent initiation of translation. Researchers typically include an IRES when cloning two or more genes and wish them to be expressed with the same promoter.

Transcription is stopped by a termination sequence that follows your gene of interest. Some examples of transcriptional terminators are:

• E. coli: T7 terminator
• Yeast: AOX and CYC1 terminators
• Insect: SV40 terminator
• Mammalian: SV40 and BGH polyA
• Viral: 3’ LTR

Epitope tags are typically included to allow for easy detection or rapid purification of your gene of interest by fusing the tag with your gene of interest. Epitope tags can be on either the N- terminus or C-terminus of your gene of interest. Here are some considerations to take into account when using an epitope tag:

1. N-terminal tags may have protease cleavage sites.
2. N-terminal tags have RBS/Kozak included.
3. Secretion signals are always N-terminal and are automatically cleaved off during Golgi processing.
4. Replace native secretion signal if present in GOI.
5. Choose an N-terminal tag when the C-terminus of the protein is important for function.
6. Ensure that a STOP codon is present at the end of your gene of interest when working with an N-terminal tag.
7. If including an N-terminal tag, ensure that your gene is in frame with the tag.
8. Choose a C-terminal tag when the N-terminus of the protein is important for function or if the protein is uncharacterized.
9. Make sure to omit the STOP codon from your gene of interest for fusion with C-terminal tags; a STOP codon is present at the end of the C-terminal tag.
10. Make sure to include your start codon (ATG) as well as a RBS/Kozak if needed at the start of your gene of interest when working with C-terminal tags.
11. If including a C-terminal tag, ensure that the tag is in frame with your gene of interest.

Here are some basic guidelines to help you select an epitope tag:

An expression vector typically contains the following elements:

• Gene of interest expression cassette (including promoter-gene-termination or poly A signal)
• Antibiotic selection cassette for particular host
• Antibiotic selection cassette for E. coli
• Bacterial origin of replication

Additional elements include:

• Cloning site
• Epitope tags
• Secretion signal (at N-terminus)

No, these vectors do not contain a functional promoter to express your gene of interest. These vectors are typically for subcloning or sequencing.

ExpressLink™ T4 DNA Ligase allows for faster ligation times and ligation at room temperature. For TA cloning, the ligation time is 15 minutes while the original T4 DNA Ligase needs a minimum of 4 hours to overnight for incubation. For blunt cloning, the ligation time with ExpressLink™ T4 DNA Ligase is reduced to 5 minutes from the 1 hour it takes with T4 DNA Ligase. The performance of these two ligases is similar, with an 80% cloning efficiency with our control PCR inserts using the above mentioned minimum ligation incubation times/temperatures.

It depends on your application. For ligation of dsDNA fragments with cohesive ends, either enzyme can be used. E. coli DNA Ligase requires the presence of β-NAD, while T4 DNA Ligase requires ATP. However, only T4 DNA Ligase can join blunt-ended DNA fragments; E. coli Ligase is unable to join such fragments.

E. coli DNA Ligase is generally used to eliminate nicks during second-strand cDNA synthesis. T4 DNA Ligase should not be substituted for E. coli DNA Ligase in second-strand synthesis because of its capability for blunt end ligation of the ds cDNA fragments, which could result in formation of chimeric inserts.

You may have to try different ratios from 1:1 to 15:1 insert:vector.

Equation:

At least one molecule in a ligase reaction (i.e., insert or vector) must be phosphorylated. Ligation reactions are dependent on the presence of a 5' phosphate on the DNA molecules. The ligation of a dephosphorylated vector with an insert generated from a restriction enzyme digest (phosphorylated) is most routinely performed. Although only one strand of the DNA ligates at a junction point, the molecule can form a stable circle, providing that the insert is large enough for hybridization to maintain the molecule in a circular form.

Recommendations vary depending on the size of the vector and insert or the nature of the insert, but for most plasmid cloning or subcloning reactions, a vector concentration of 1-10 ng/µl is recommended. Inserts should generally be 2- to 3-fold excess in molar concentration relative to the vector.

If working with a vector that contains the lac promoter and the LacZ α fragment (for α complementation), blue/white screening can be used as a tool to select for presence of the insert. X-gal is added to the plate as a substrate for the LacZ enzyme and must always be present for blue/white screening. The minimum insert size needed to completely disrupt the LacZ gene is 400 bp. If the LacIq repressor is present (either provided by the host cells, for example TOP10F', or expressed from the plasmid) it will repress expression from the lac promoter, thus preventing blue/white screening. Hence in the presence of the LacIq repressor, IPTG must be provided to inhibit the LacIq. Inhibition of LacIq permits expression from the lac promoter for blue/white screening. X-gal (also known as 5-bromo-4-chloro-3-indolyl β-D-glucopyranoside) is soluble in DMSO or DMF, and can be stored in solution in the freezer for up to 6 months. Protect the solution from light. Final concentration of X-gal and IPTG in agar plates: Prior to pouring plates, add X-gal to 20 mg/mL and IPTG to 0.1 mM to the medium. When adding directly on the surface of the plate, add 40 µl X-gal (20 mg/mL stock) and 4 µl IPTG (200 mg/mL stock).

TOPO® vectors containing the LacZ-ccdB cassette allow direct selection of recombinants via disruption of the lethal E. coli gene, ccdB. Ligation of a PCR product disrupts expression of the LacZ-ccdB gene fusion permitting growth of only positive recombinants upon transformation. Cells that contain non-recombinant vector are killed upon plating. Therefore, blue/white screening is not required. When doing blue/white color screening of clones in TOPO® vectors containing the LacZ-ccdB cassette, colonies showing different shades of blue may be observed. It is our experience that those colonies that are light blue as well as those that are white generally contain inserts. The light blue is most likely due to some transcription initiation in the presence of the insert for the production of the lacZ α without enough ccdB expressed to kill the cells and is insert dependent. To completely interrupt the lacZ gene, inserts must be >400 bp; therefore an insert of 300 bp can produce a light blue colony. A white colony that does not contain an insert is generally due to a spontaneous mutation in the ccdB gene.

A minimum insertion of 150 bp is needed in order to ensure disruption of the ccdB gene and prevent cell death. (Reference: Bernard et al., 1994. Positive-selection vectors using the F plasmid ccdB killer gene. Gene 148: 71-74.)

Strains that contain an F plasmid, such as TOP10F’, are not recommended for transformation and selection of recombinant clones in any TOPO® vector containing the ccdB gene. The F plasmid encodes the ccdA protein, which acts as an inhibitor of the ccdB gyrase-toxin protein. The ccdB gene is also found in the ccd (control of cell death) locus on the F plasmid. This locus contains two genes, ccdA and ccdB, which encode proteins of 72 and 101 amino acids respectively. The ccd locus participates in stable maintenance of F plasmid by post-segregational killing of cells that do not contain the F plasmid. The ccdB protein is a potent cell-killing protein when the ccdA protein does not inhibit its action.

If working with a vector that contains the lac promoter and the LacZ α fragment (for α complementation), blue/white screening can be used as a tool to select for presence of the insert. X-gal is added to the plate as a substrate for the LacZ enzyme and must always be present for blue/white screening. The minimum insert size needed to completely disrupt the lacZ gene is 400 bp. If the LacIq repressor is present (either provided by the host cells, for example TOP10F', or expressed from the plasmid), it will repress expression from the lac promoter thus preventing blue/white screening. Hence, in the presence of the LacIq repressor, IPTG must be provided to inhibit the LacIq. Inhibition of LacIq permits expression from the lac promoter for blue/white screening.