Directional Topo^® Cloning

How Topoisomerase I Works

Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5´ -CCCTT in one strand (Shuman, 1991). 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 (Shuman, 1994). TOPO^® Cloning exploits this reaction to efficiently clone PCR products (see diagram below).

The design of the PCR primers to amplify your gene of interest is critical for expression. Depending on the pET TOPO^® vector you are using, consider the following when designing your PCR primers:

• Sequences required to facilitate directional cloning (see below)
• Whether or not you wish to clone your PCR product in frame with the appropriate N-terminal and/or C-terminal peptide tag 

General Requirements for the Forward Primer

To enable directional cloning, the forward PCR primer must contain the sequence, CACC, at the 5´ end of the primer. The 4 nucleotides, CACC, base pair with the overhang sequence, GTGG, in each pET TOPO^® vector.
 
For example, below is the DNA sequence of the N-terminus of a theoretical protein and the proposed sequence for your forward PCR primer:
 
DNA sequence:                         5´  -ATG GGA TCT GAT AAA
 
Proposed Forward PCR primer: 5´ -C ACC ATG GGA TCT GAT AAA

General Requirements for the Reverse Primer

In general, design the reverse PCR primer to allow you to clone your PCR product in frame with any C-terminal tag, if desired. To ensure that your PCR product clones directionally with high efficiency, the reverse PCR primer must not be complementary to the overhang sequence GTGG at the 5´ end. A one base pair mismatch can reduce the directional cloning efficiency from 90% to 75%, and may increase the chances of your ORF cloning in the opposite orientation. We have not observed evidence of PCR products cloning in the opposite orientation from a two base pair mismatch, but this has not been tested thoroughly.
 
Example: Below is the sequence of the C-terminus of a theoretical protein. You want to clone in frame with the C-terminal tag. The stop codon is underlined.
 
DNA sequence:                        AAG TCG GAG CAC TCG ACG ACGGTG tag-3´
 
One solution is to design the reverse PCR primer to start with the codon just up-stream of the stop codon, but the last two codons contain GTGG (underlined below), which is identical to the overhang sequence. As a result, the reverse primer will be complementary to the overhang sequence, increasing the probability that the PCR product will clone in the opposite orientation. You want to avoid this situation.
 
DNA sequence:                       AAG TCG GAG CAC TCG ACG ACG GTG tag-3´
Proposed Reverse PCR primer sequence:      TG AGC TGC TGC CAC-5´
 
Another solution is to design the reverse primer so that it hybridizes just down-stream of the stop codon, but still includes the C-terminus of the ORF. Note that you will need to replace the stop codon with a codon for an innocuous amino acid such as glycine or alanine.

• Remember that the pET TOPO^® vectors accept blunt-end PCR products. Refer below for a discussion of specific factors to consider when designing PCR primers for cloning into each pET TOPO^® vector.
• Do not add 5´ phosphates to your primers for PCR. This will prevent ligation into the pET TOPO^® vectors.
• We recommend gel-purifying your oligonucleotides, especially if they are long (> 30 nucleotides).

Example of Primer Design

The example below uses a theoretical protein and is for illustration purposes only. In this case, PCR primers are designed to allow cloning of the PCR product into pET101/D-TOPO^® . In this example, the N-terminus of the protein is encoded by:

 5´ -atggcccccccgaccgatgtcagcctgggggacgaa…

1. Design the forward PCR primer to be:

 5´ -caccatggcccccccgaccgat-3´



2. For the reverse primer, analyze the C-terminus of the protein.
   
   …GCG  GTT AAG TCG GAG CAC TCG ACG ACT GCA TAG-3´
   
   …CGC CAA TTC AGC CTC GTG AGC TGC TGA CGT   ATC-5´


3. To fuse the ORF in frame with the V5 epitope and 6xHis tag, remove the stop codon by starting with nucleotides homologous to the last codon (TGC) and continue upstream (underlined sequence in the bottom strand above). The reverse primer will be:
   
       5´ -TGC AGT CGT CGA GTG CTC CGA CTT-3´


4. This will amplify the C-terminus without the stop codon and allow you to clone the ORF in frame with the V5 epitope and 6xHis tag. If you don’t want the V5 epitope and 6xHis tag, simply begin with the stop codon:
   
     5´ -CTA TGC AGT CGT CGA GTG CTC CGA CTT-3´ 
    
   TOP

Introduction
 
Pfx50™ DNA Polymerase is a fusion enzyme consisting of recombinant DNA polymerase from the archaean Thermococcus zilligii fused to an accessory protein. The highly thermostable polymerase possesses a proofreading 3’ → 5’ exonuclease activity, while the accessory protein stabilizes primer-template complexes in PCR.
 
Pfx50™ DNA Polymerase offers 50 times better fidelity than Taq DNA polymerase,coupled with high specificity and an extremely fast elongation rate (as fast as 15 seconds per kb). In addition, the fusion enzyme has an intrinsic hot-start capability for room-temperature reaction assembly.
 
Note:

• 10X Pfx50™ PCR buffer contains BSA; store at –20ºC.
•  Pfx50™ DNA Polymerase produces blunt-end PCR products, which can be used with Directional TOPO^® Cloning and Zero Blunt^® TOPO^® Cloning technologies. 

Component	100 Rxn Kit	500 Rxn Kit
Pfx50™ DNA Polymerase (5 U/µl)	100 µl	500 µl
10X Pfx50™ PCR Mix	1.3 ml	2 × 1.3 ml
50-mM Magnesium Sulfate	1 ml	1 ml

Pfx50™ DNA Polymerase Storage Buffer

20 mM Tris-HCl (pH 8.0), 40 mM KCl, 0.1 mM EDTA, 1 mM DTT, stabilizers, and 50% (v/v) glycerol
 
Unit Definition

One unit of Pfx50™ DNA Polymerase incorporates 10 nmol of deoxyribonucleotide into acid-insoluble material in 30 min at 74°C.
 
General Recommendations and Guidelines for PCR

General PCR parameters and troubleshooting information are documented in Innis, et al.
 
Template:  Pfx50™ DNA Polymerase is suitable for amplifying targets up to 4 kb from the following templates:

Template                    Amount
Genomic DNA             1–200 ng
Plasmid DNA              1–100 pg
cDNA                          3­–5 µl from 10 ng to 1 µg starting total RNA

Amplification of longer targets (up to 7 kb) may be possible, but may require more template and longer elongation times.
 
Primers:  Use 0.3 µM per primer as a general starting point. For larger amounts of template (e.g., 200 ng genomic DNA), increasing the concentration up to 0.5 µM per primer may improve yield.
 
Annealing Temperature:  The annealing temperature is slightly higher than with typical PCR. The optimal annealing temperature should be ~2ºC lower than the Tm of the primers used. A range of 60–68ºC is recommended.
 
MgSO₄ :  MgSO₄ is included in the 10X Pfx50™ PCR Mix at a final concentration of 1.2 mM, which is sufficient for most templates. For further optimization, add 0.1 µl to 1.0 µl of 50-mM MgSO₄ .
 
Extension Time:  As little as 15 seconds per kb may be used; 30 seconds per kb is suitable for most targets. Use up to 60 seconds per kb for maximum yield.
 
Protocol
 
The following procedure is suggested as a starting point when using Pfx50™ DNA Polymerase in any PCR amplification.
 

1. Program the thermal cycler as follows (see the note on annealing temperature above):
 
Initial denaturation:      94ºC for 2 minutes
35 cycles of:
Denaturation:                94ºC for 15 seconds
Annealing:                     60–68ºC (Tm of primers minus 2ºC) for 10–30 seconds
Extension:                     68ºC for 30–60 seconds per kb of PCR product
Final extension:            68ºC for 5 minutes
 


2. Add the following components to an autoclaved microcentrifuge tube at room temperature (for multiple reactions, prepare a Master Mix of common components to enable accurate pipetting):
   
   
   

   Component	Volume	Final Conc.
   10X Pfx50™ PCR Mix	5 µl	1X
   10 mM dNTP Mix	1.5 µl	0.3 mM each
   Primer mix (10 µM each)	1.5 µl	0.3 µM each
   Template DNA ≥1 µl	As required
   Pfx50™ DNA Polymerase (5 U/µl)	1 µl	5 units
   Autoclaved, distilled water to	50 µl

    


3. Cap the tube, tap gently to mix, and centrifuge briefly to collect the contents.


4. Place the tube in the thermal cycler and run the program from Step 1. After cycling, maintain the reaction at 4ºC. Samples can be stored at –20ºC until use.

Analyze products using E-Gel^® Pre-Cast agarose gels or standard agarose gel electrophoresis. Visualize by staining with SYBR Safe™ DNA gel stain or ethidium bromide.

Introduction

Smearing, multiple banding, primer-dimer artifacts, or large PCR products (>3 kb) may necessitate gel purification. If you intend to purify your PCR product, be extremely careful to remove all sources of nuclease contamination. There are many protocols to isolate DNA fragments or remove oligonucleotides. Refer to Current Protocols in Molecular Biology, Unit 2.6 (Ausubel et al., 1994) for the most common protocols. Three simple protocols are provided below.  
 
Cloning efficiency may decrease with purification of the PCR product. You may wish to optimize your PCR to produce a single band.
 
Using the S.N.A.P.™ Gel Purification Kit

The S.N.A.P.™ Gel Purification Kit (Catalog no. K1999-25) allows you to rapidly purify PCR products from regular agarose gels.
 

1.  Electrophorese amplification reaction on a 1 to 5% regular TAE agarose gel.
 
Note:   Do not use TBE to prepare agarose gels. Borate interferes with the sodium iodide step, below.
 


2. Cut out the gel slice containing the PCR product and melt it at 65°C in 2 volumes of the 6 M sodium iodide solution.


3. Add 1.5 volumes Binding Buffer


4. Load solution (no more than 1 ml at a time) from Step 3 onto a S.N.A.P.™ column. Centrifuge 1 minute at 3000 x g in a microcentrifuge and discard the supernatant.


5. If you have solution remaining from Step 3, repeat Step 4.


6. Add 900 µl of the Final Wash Buffer.


7. Centrifuge 1 minute at full speed in a microcentrifuge and discard the flow-through.


8. Repeat Step 7.


9. Elute the purified PCR product in 40 µl of TE or sterile water. Use 4 µl for the TOPO^® Cloning reaction.

 
Quick S.N.A.P.™ Method

An even easier method is to simply cut out the gel slice containing your PCR product, place it on top of the S.N.A.P.™ column bed, and centrifuge at full speed for 10 seconds. Use 1-2 µl of the flow-through in the TOPO^® Cloning reactionpage 18). Be sure to make the gel slice as small as possible for best results.

Low-Melt Agarose Method
 
If you prefer to use low-melt agarose, use the procedure below. Note that gel purification will result in dilution of your PCR product and a potential loss of cloning efficiency.
 
  1.     Electrophorese as much as possible of your PCR reaction on a low-melt agarose gel (0.8 to 1.2%) in TAE buffer.
 
  2.     Visualize the band of interest and excise the band.
 
  3.     Place the gel slice in a microcentrifuge tube and incubate the tube at 65°C until the gel slice melts.
 
  4.     Place the tube at 37°C to keep the agarose melted.
 
  5.     Add 4 µl of the melted agarose containing your PCR product to the TOPO^® Cloning reaction.
 
  6.     Incubate the TOPO^® Cloning reaction at 37°C for 5 to 10 minutes. This is to keep the agarose melted.
 
  7.     Transform 2 to 4 µl directly into One Shot^®TOP10 cells.
 
The cloning efficiency may decrease with purification of the PCR product. You may wish to optimize your PCR to produce a single band.

Introduction

Once you have produced the desired PCR product, you are ready to TOPO^®Clone it into the pET TOPO^® vector and transform the recombinant vector into One Shot^® TOP10 E. coli. You should have everything you need set up and ready to use to ensure that you obtain the best possible results. We suggest that you read the this section and the next section entitled Transforming Chemically Competent Cells before beginning. If this is the first time you have TOPO^® Cloned, perform the control reactions (see Performing Control Reactions) in parallel with your samples. 

Amount of PCR Product to Use in the TOPO^® Cloning Reaction

When performing directional TOPO^® Cloning, we have found that the molar ratio of PCR product:TOPO^® vector used in the reaction is critical to its success. To obtain the highest TOPO^®Cloning efficiency, use a 0.5:1 to 2:1 molar ratio of PCR product:TOPO^® vector.

Note that the TOPO^® Cloning efficiency decreases significantly if the ratio of PCR product: TOPO^® vector is <0.1:1 or >5:1  These results are generally obtained if too little PCR product is used (i.e. PCR product is too dilute) or if too much PCR product is used in the TOPO^®Cloning reaction. If you have quantitated the yield of your PCR product, you may need to adjust the concentration of your PCR product before proceeding to TOPO^® Cloning.

Tip:   For the pET TOPO^® vectors, using 1-5 ng of a 1 kb PCR product or 5-10 ng of a 2 kb PCR product in a TOPO^® Cloning reaction generally results in a suitable number of colonies.



Using Salt Solution in the TOPO^® Cloning Reaction

You will perform TOPO^® Cloning in a reaction buffer containing salt (i.e. using the stock salt solution provided in the kit). Note that the amount of salt added to the TOPO^® Cloning reaction varies depending on whether you plan to transform chemically competent cells (provided) or electrocompetent cells. 

• If you are transforming chemically competent E. coli, use the stock Salt Solution as supplied and set up the TOPO^® Cloning reaction as directed below.
• If you are transforming electrocompetent E. coli, the amount of salt in the TOPO^® Cloning reaction must be reduced to 50 mM NaCl, 2.5 mM MgCl₂ to prevent arcing during electroporation. Dilute the stock Salt Solution 4-fold with water to prepare a 300 mM NaCl, 15 mM MgCl₂  Dilute Salt Solution. Use the Dilute Salt Solution to set up the TOPO ^® Cloning reaction as directed below. 

Performing the TOPO^® Cloning Reaction

Use the procedure below to perform the TOPO^® Cloning reaction. Set up the TOPO^®Cloning reaction depending on whether you plan to transform chemically competent E. coli or electrocompetent E. coli. Reminder: For optimal results, be sure to use a 0.5:1 to 2:1 molar ratio of PCR product:TOPO^® vector in your TOPO^® Cloning reaction.
 
Note: The blue color of the TOPO^® vector solution is normal and is used to visualize the solution

Reagents*	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 (1:4)	--	1 µl
Sterile Water	add to a final volume of 5 µl	add to a final volume of 5 µl
TOPO® vector	1 µl	1 µl
Total Volume	6 µl	6 µl

*Store all reagents at -20°C when finished. Salt solution and water can be stored at room temperature or +4°C.
 
  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^® 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 may yield more colonies.
 
  2.     Place the reaction on ice and proceed to Transforming Chemically Competent Cells.

Note:   You may store the TOPO^®Cloning reaction at -20°C overnight.

1. Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. S. (eds) (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, CA