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TOPO TA Cloning for Sequencing Protocol

Five-minute cloning of Taq polymerase-amplified PCR Products for sequencing

 

Principle:

 

Introduction:

The TOPO TA Cloning Kits for Sequencing provide a highly efficient, 5-minute, one-step cloning strategy ("TOPO Cloning") for the direct insertion of Taq polymerase-amplified PCR products into a plasmid vector for sequencing. No ligase, post-PCR procedures, or PCR primers containing specific sequences are required. Storage: Store Box 1 at -20 °C and Box 2 at -80 °C.

 

How It Works:

 

The plasmid vector (pCR 4-TOPO) supplied is linearized with:

•  single 3’ thymidine (T) overhangs for TA Cloning

•  topoisomerase covalently bound to the vector (referred to as "activated"

vector)

 

Taq polymerase has a non-template-dependent terminal transferase activity,

which adds a single deoxyadenosine (A) to the 3’ ends of PCR products. The linearized vector supplied in this kit has single, overhanging 3’ deoxythymidine  (T) residues. This allows PCR inserts to ligate efficiently with the vector.

 

Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5-CCCTT in one strand. The energy from the broken phosphodiester backbone is conserved by

formation of a covalent bond between the 3 phosphate of the cleaved strand and a tyrosyl residue (Tyr-274) of topoisomerase I. The phospho-tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5 hydroxyl of the original cleaved strand, reversing the reaction and releasing topoisomerase. TOPO Cloning exploits this reaction to efficiently clone PCR products.

 

Experimental Outline:

The flow chart below outlines the experimental steps necessary to clone a PCR product.

  • Produce PCR product
  • TOPO-Cloning Reaction:
  • Mix together PCR product and pCR 4-TOPO
  • Incubate 5 minutes                                                 at room temperature
  • Transform into TOPO10 E.coli cells
  • Select and analyze colonies
  • Isolate plasmid DNA and sequence

Producing PCR Products

The TOPO TA Cloning Æ Kits for Sequencing are specifically designed to clone Taq polymerase-generated PCR products for sequencing. The first time you use the kit, we recommend performing the control TOPO Cloning reaction on page 13 to evaluate your results. Do not add 5’ phosphates to your primers for PCR. The PCR product synthesized will not ligate into pCR 4-TOPO.

 

Materials Supplied by the User

In addition to general microbiological supplies  (e.g. plates, spreaders), you will need the following reagents and equipment:

•  Taq polymerase

•  Thermocycler

•  DNA template and primers for PCR product

 

Polymerase Mixtures

 

1. Set up the following 50 µl PCR reaction. Use the cycling parameters suitable for your primers and template. Be sure to end with a 7-30 minute extension at 72°C to ensure that all PCR products are full length and 3’adenylated. Use less DNA for plasmid template DNA and more DNA for genomic template DNA.

DNA Template                        10-100 ng

10X PCR Buffer                      5 µl

50 mM dNTPs                         0.5 µl

Primers (~200 ng each)            1 µM each

Sterile water                            add to a final volume of 49 µl

Taq Polymerase (1 unit/µl)        1 unit

____________________________________________________

Total Volume 50 µl

 

2. Check the PCR product by agarose gel electrophoresis. You should see a

single, discrete band. If you do not obtain a single, discrete band from your PCR, you may gel-purify your fragment before TOPO Cloning. Take special care to avoid sources of nuclease contamination. Alternatively, you may elect to optimize your PCR to eliminate multiple bands and smearing

Setting Up the TOPO Cloning Reaction

 

The table below describes how to set up your TOPO  Cloning reaction (6 µl) for eventual transformation into either chemically competent or electrocompetent TOP10 E. coli. Additional information on optimizing the TOPO Cloning reaction for your needs can be found on page 11.

Note: The red or yellow color of the TOPO Æ vector solution is normal and is used to visualize the solution.

___________________________________________________________

Reagent*          Chemically Competent E. coli      Electrocompetent E. coli

___________________________________________________________

Fresh PCR            product 0.5 to 4 µl                        0.5 to 4 µl

Salt Solution                   1 µl --

Dilute Salt Solution      -- 1 µl

Sterile Water        final volume of 5 µl                   final volume of 5 µl

TOPO vector                  1 µl                                         1 µl

__________________________________________________________

*Store all reagents at -20°C when finished. Salt solutions and water can be stored at room temperature or +4°C.

 

Performing the TOPO Cloning Reaction:

 

  1. Mix reaction gently and incubate for 5 minutes at room temperature (22-23°C). Note: For most applications, 5 minutes will yield plenty of colonies for analysis. Depending on your needs, the length of the TOPO Æ Cloning reaction can be varied from 30 seconds to 30 minutes. For routine subcloning of PCR products, 30 seconds may be sufficient. For large PCR products (> 1 kb) or if you are TOPO Cloning a pool of PCR products, increasing the reaction time will yield more colonies.
  2. Place the reaction on ice and proceed to Transforming One Shot Æ Competent Cells (next page). Note: You may store the TOPO Cloning reaction at -20°C overnight.

 

Chemical Transformation Protocol

 

This protocol is only recommended for transformations using ampicillin selection. Note: It is essential that LB plates containing ampicillin are pre-warmed prior to spreading.

 

  1. Add 2 µl of the TOPO Cloning reaction (from “Performing the TOPO Cloning Reaction, Step 2”) into a vial of One Shot Chemically Competent E. coli and mix gently. Do not mix by pipeting up and down.
  2. Incubate on ice for 5 minutes. Note: Longer incubations on ice do not seem to have any affect on transformation efficiency.
  3. Heat-shock the cells for 30 seconds at 42°C without shaking.
  4. Immediately transfer the tube to ice.
  5. Add 250 µl of room temperature SOC medium.
  6. Cap the tube tightly and shake the tube horizontally (200 rpm) at 37 °C for 1 hour.
  7. Spread 50 µl of cells on a pre-warmed LB plate containing 50-100 µg/ml ampicillin and incubate overnight at 37°C.
  8. An efficient TOPO Cloning reaction should produce several hundred colonies. Pick ~10 colonies for analysis.

 

One Shot Electroporation

 

  1. Add 2 µl of the TOPO Cloning reaction to 50 µl One Shot TOP10 Electrocomp  E. coli in a 0.1 cm cuvette and mix gently. Do not mix by pipetting up and down.
  2. Electroporate your samples using your own protocol and your electroporator. Note: If you have problems with arcing, see below.
  3. Add 250 µl of room temperature SOC medium.
  4. Transfer the solution to a 15 ml snap-cap tube (e.g. Falcon) and shake for at least 1 hour at 37ºC to allow expression of the antibiotic resistance genes.
  5. Spread 10-50 µl from each transformation onto a pre-warmed selective plate and incubate overnight at 37°C. To ensure even spreading of small volumes, add 20 µl of SOC. We recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies.
  6. An efficient TOPO Cloning reaction should produce hundreds of colonies. Pick ~10 colonies for analysis.

 

References

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994). Current Protocols in Molecular Biology (New York: Greene Publishing Associates and Wiley-Interscience).

Bernard, P., and Couturier, M. (1992). Cell Killing by the F Plasmid CcdB Protein Involves Poisoning of DNA-Topoisomerase II Complexes. J. Mol. Biol. 226, 735-745. Bernard, P., Gabant, P., Bahassi, E. M., and Couturier, M. (1994). Positive Selection Vectors Using the F Plasmid ccdB Killer Gene. Gene 148, 71 74.

Bernard, P., Kezdy, K. E., Melderen, L. V., Steyaert, J., Wyns, L., Pato, M. L., Higgins, P. N., and Couturier, M. (1993). The F Plasmid CcdB Protein Induces Efficient ATP-dependent DNA Cleavage by Gyrase. J. Mol. Biol. 234, 534-541.

Brownstein, M. J., Carpten, J. D., and Smith, J. R. (1996). Modulation of Non-Templated Nucleotide Addition by Taq DNA Polymerase: Primer Modifications that Facilitate Genotyping. BioTechniques 20, 1004-1010.

Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. S. (1990) PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989).

Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).

Shuman, S. (1994). Novel Approach to Molecular Cloning and Polynucleotide Synthesis Using Vaccinia DNA Topoisomerase. J. Biol. Chem. 269, 32678-32684.

Shuman, S. (1991). Recombination Mediated by Vaccinia Virus DNA Topoisomerase I in Escherichia coli is Sequence Specific. Proc. Natl. Acad. Sci. USA 88, 10104-10108.