PCR Cloning Considerations

Nature of the ｉｎｓｅｒｔ

The cloning of PCR-amplified fragments into a linear vector is typically a rapid and efficient process. However, not all PCR fragments will clone with the same efficiency into the same vector. These differences may be due to fragment size, ｉｎｓｅｒｔ toxicity, and the complexity of the ｉｎｓｅｒｔ. Inverted, AT-rich, or GC-rich repeats may contribute to the instability of the fragment as a cloned product in any vector(pCR®II, pCR®2.1, pcDNA™3.1, pUC18).

ｉｎｓｅｒｔ Size

The size of the fragment being cloned is a primary contributor to the overall cloning efficiency. Large fragments of DNA (≥ 5 kb) are amenable to cloning in high-copy number vectors, yet at a much lower efficiency.

Vector-to-ｉｎｓｅｒｔ Ratio

Optimization of molar concentration ratios of the vector to ｉｎｓｅｒｔ is critical to ensure efficient cloning. Successful cloning ratios may range from 1:1 to 1:10. One common strategy for determining the optimal ratio is by preparing several vector: ｉｎｓｅｒｔ ratios: 1:1, 1:3, and 1:5. While these ratios may not be ideal for all cloning events, they are useful for most cloning needs. For example, if the vector is 3 kb and the ｉｎｓｅｒｔ is 1 kb, one-third the amount of ｉｎｓｅｒｔ needs to be added to attain a 1:1 molar ratio. When performing TOPO® -TA or Directional TOPO® Cloning, optimal results are achieved most often when using a 1:10 dilution of the PCR product.

Fresh PCR Product

The use of fresh PCR products in TA, TOPO® TA, and Directional TOPO® Cloning is recommended due the potential presence of exonucleases that will, over time, degrade the nucleotide overhangs, reducing the efficiency of the cloning event. While it is not recommended, some PCR products have been successfully cloned after 1 week of storage at +4℃.

Importance of Positive and Negative Controls

In any cloning experiment, the use of positive and negative controls is important. Without appropriate positive and negative controls for your cloning and transformation reactions, it is very difficult to evaluate the results of a cloning event. These controls are indicators of enzyme activity in DNA preparation and transformation efficiency of competent cells. Troubleshooting is virtually impossible without any controls. To ensure the efficiency of the cloning reaction, each of Invitrogen’s kits includes controls.

Compatibility of DNA Ends of Vector and ｉｎｓｅｒｔ

TA Cloning® technology was designed to clone PCR products produced by Taq polymerase. It takes advantage of the terminal transferase activity of this polymerase which adds a single 3’-A overhang to each end of the PCR product. Blunt cloning vectors and directional TOPO® cloning technologies are designed to clone PCR products produced by proofreading polymerases. Successful cloning depends upon using the correct polymerase with your cloning vector.

Addition of 3’-A Overhangs Following PCR Amplification

Direct cloning of DNA amplified by proofreading polymerases into TA Cloning® or TOPO TA Cloning® vectors is often difficult because of very low cloning efficiencies. This is because proofreading polymerases possess 3´→5´ exonuclease activity that removes the 3´-A overhangs necessary for TA Cloning® and TOPO TA Cloning®. A simple procedure to add 3´ adenines to blunt-end fragments is provided below. Other protocols may be suitable.

You will need the following items:

• Taq polymerase

• A heat block equilibrated to 72°C

• Phenol-chloroform

• 3 M sodium acetate

• 100% ethanol

• 80% ethanol

• TE buffer