Invitrogen™ Zero Blunt™ TOPO PCR Cloning Kit for Subcloning

Catalog No.	Quantity
K283020	25 Reactions

Catalog No. K283020 Supplier Invitrogen™ Supplier No. K283020

Description

Includes

Linearized and topoisomerase I-activated pCR™Blunt II-TOPO™ vector; Salt solution; dNTPs; Control template; M13 forward and reverse primers; Sterile water; One Shot™ Chemically Competent or Electrocomp™ E. coli; S.O.C. medium; Supercoiled pUC19 control plasmid

Combines the unique Zero Background™ technology with the pCR™- Blunt II-TOPO vector to allow easy, high-efficiency cloning of blunt-end PCR products

• Unique technology to minimize background
• Get your clone: 95% efficiency
• Multiple primer sites (T7, T3, M13F, M13R) make sequence analysis or PCR easy and convenient
• Kanamycin and Zeocin resistance genes for your choice of selection
• Convenient, validated restriction sites in MCS for further subcloning
• Includes MachI™ T1R competent cells for fast miniprep and shortens cloning time

Chromatin Biology, Chromatin Immunoprecipitation (ChIP), Cloning, Methylation Analysis, PCR Cloning, RNAi, Epigenetics and Non-Coding RNA Research, Sequencing Bisulfite-Treated DNA

Specifications

• Bacterial or Yeast Strain: Mach1™
• Cloning Method: Blunt TOPO™
• Content And Storage: Box 1:
  • Linearized and topoisomerase I-activated pCR™Blunt II-TOPO™ vector
  • Salt solution
  • dNTPs
  • Control template
  • M13 forward and reverse primers
  • Sterile water
  
  Store at -5 to -30°C.
  All reagents are stable for 6 months when properly stored.
  
  Box 2:
  • One Shot™ Chemically Competent or Electrocomp™ E. coli
  • S.O.C. medium
  • Supercoiled pUC19 control plasmid
  
  Store at -68 to -85°C.
• Format: Kit
• For Use With (Application): Chromatin Biology
• Product Line: One Shot
• Product Type: PCR Cloning Kit
• Quantity: 25 Reactions
• Vector: Blunt DNA Cloning Vectors

Frequently Asked Questions (FAQs)

What is the best molar ratio of PCR product:vector to use for TOPO TA cloning? Is there an equation to calculate the quantity to use?

We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1. The quantity used in a TOPO cloning reaction is typically 5-10 ng of a 2 kb PCR product.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 (insert:vector ratio)

What is the best ratio of insert:vector to use for cloning? Is there an equation to calculate this?

The optimal ratio is 1:1 insert to vector. Optimization can be done using a ratio of 0.5-2 molecules of insert for every molecule of the vector.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio

I'm seeing a lot of vector-only colonies when I try to perform a negative control reaction using vector only (no insert) for a TOPO reaction. Is my TOPO vector re-ligating?

Using the vector only for transformation is not a recommended negative control. The process of TOPO-adaptation is not a 100% process, therefore, there will be “vector only” present in your mix, and colonies will be obtained.

I'm trying to clone in my phosphorylated PCR product into a TOPO vector, and I'm getting no colonies. However, when I clone the same product into a TA vector, everything works perfectly. Why is this?

Phosphorylated products can be TA cloned but not TOPO cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. TOPO vectors have a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. Non-TOPO linear vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO cloning.

I'm able to get a lot of colonies, however, none contain my insert of interest. What should I do?

You may be cloning in an artifact. TA and TOPO Cloning are very efficient for small fragments (< 100 bp) present in certain PCR reactions. Gel-purify your PCR product using either a silica-based DNA purification system or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)

A majority of colonies are blue or light blue, with very few white colonies. What should I do?

There could be a few possibilities for this:

- The insert does not interrupt the reading frame of the lacZ gene. If you have a small insert (< 500 bp), you may have light blue colonies. Analyze some of these blue colonies as they may contain insert.
- A polymerase that does not add 3' A-overhangs was used. If you used a proofreading enzyme, you will need to do a post-reaction treatment with Taq polymerase to add the 3' A-overhangs.
- PCR products were gel-purified before ligation. Gel purification can remove the single 3' A- overhangs. Otherwise, optimization of your PCR can be performed so that you can go directly from PCR to cloning.
- The PCR products were stored for a long period of time before ligation reaction. Use fresh PCR products. Efficiencies are reduced after as little as 1 day of storage.
- Too much of the amplification reaction was added to the ligation. The high salt content of PCR can inhibit ligation. Use no more than 2-3 µl of the PCR mixture in the ligation reaction.
- The molar ratio of vector:insert in the ligation reaction may be incorrect. Estimate the concentration of the PCR product. Set up the ligation reaction with a 1:1 or 1:3 vector:insert molar ratio.
On a typical plate there are a few white colonies which do not contain insert. These are usually larger than the other colonies and are due to a deletion of a portion of the plasmid sequence by a rare recombination event (usually from the polylinker to a site in the F1 origin). To find a colony with an insert it is best to pick clones of a variety of color and pattern for analysis. Often an insert will generate two distinct patterns according to its orientation.

I'm getting no colonies after transformation. What should I do?

No colonies may occur due to the following problems:

Bacteria were not competent. Use the pUC18 vector included with the One Shot module to check the transformation efficiency of the cells.
- Incorrect concentration of antibiotic on plates, or the plates are too old. Use 100 µg/mL of ampicillin or 50 µg/mL kanamycin. Be sure ampicillin plates are fresh (< 1 month old).
- The product was phosphorylated (TOPO cloning only). Phosphorylated products can be TA-cloned but not TOPO-cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. The TOPO vector has a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. The non- TOPO vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO-cloning.

I'm getting low cloning efficiency with my TOPO cloning reactions. What should I do?

Please consider the following possible causes:
- pH > 9: Check the pH of the PCR amplification reaction and adjust with 1 M Tris-HCl, pH 8.
- Excess (or overly dilute) PCR product: Reduce (or concentrate) the amount of PCR product.
- Incomplete extension during PCR: Be sure to include a final extension step of 7 to 30 minutes during PCR. Longer PCR products will need a longer extension time.
- Cloning large inserts (>1 kb): Try one or all of the following suggestions: Increase amount of insert. Incubate the TOPO cloning reaction longer. Gel-purify the insert using either a silica-based DNA purification system (e.g., PureLink system) or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)
- PCR product does not contain sufficient 3' A-overhangs even though you used Taq polymerase: Increase the final extension time to ensure all 3' ends are adenylated. Taq polymerase is less efficient at adding a nontemplate 3' A next to another A. Taq is most efficient at adding a nontemplate 3' A next to a C. You may have to redesign your primers so that they contain a 5' G instead of a 5´ T.

I'm getting very few colonies after transformation of my TOPO cloning reaction. How can I increase the number of primary colonies?

Please try the suggestions below to increase the number of colonies.
- Longer incubation of the TOPO cloning reaction at room temperature, provided that the 6X Salt solution is added to the reaction.
- Electroporation can give significant increases in colony numbers; often 10-20 fold higher. However, if doing electroporation, it is important that the TOPO reaction mix contains diluted Salt solution or, for best results, precipitated prior to transformation. For high primary transformants by electroporation it is recommended to:
- Add 100 µL double diH2O to the 6 µL TOPO reaction and incubate 10 more minutes at 37 degrees C.
- Precipitate by adding 10 µL 3 M Na-Acetate, 2 µL 20 µg/µL glycogen, 300 µL 100% ethanol. Place on dry ice or –80 degrees C for 20 min, spin at top speed in a microcentrifuge at 4 degrees C for 15 min. Wash pellet with 800 µL 80% ethanol, spin at top speed for 10 min, pour off ethanol, spin 1 min, and remove remaining ethanol without disturbing pellet. Dry pellet (air-dry or speed-vac).
- Resuspend pellet in 10 µL ddH2O and electroporate 3.3 µL of resuspended DNA according to a normal electroporation protocol. This electroporation protocol can yield up to 20 fold more colonies than chemical transformation of an equivalent TOPO-reaction. The addition of the 100 µL ddH2O followed by 10 mins incubation is not absolutely necessary, but it sufficiently dilutes the reaction and may help inactivate topoisomerase so that it is more easily electroporated.

I'm planning on cloning a 1kb fragment for sequencing and want to minimize the amount of vector sequence in my data. Which of your vectors should I use?

We would suggest using our TOPO TA cloning kit for sequencing, which contains the pCR 4 TOPO vector, or our Zero Blunt TOPO PCR cloning kit for sequencing, which contains the pCR4Blunt-TOPO vector.

I have a blunt-ended restriction product cut from another cloning vector. Can I insert this into a Zero Blunt TOPO vector?

Yes, though you will need to treat it with calf intestinal phosphatase (CIP) to get rid of the 5' phosphate group for TOPO Cloning.

Which PCR polymerases do you recommend for TA/Blunt/D-TOPO cloning and why?

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.

What PCR enzyme would you recommend for use with the Directional TOPO Cloning Kits?

For the Directional TOPO Cloning Vectors, a PCR product must be generated by a proofreading enzyme to create a blunt product. Pfx50 or Accuprime Pfx and Accuprime Pfx Supermix from Thermo Fisher Scientific are recommended for use.

When cloning a Pfx-amplified PCR product, the insert to vector ratio is an important consideration. The PCR product generally needs to be diluted since Pfx generates a high concentration of product and using too much insert DNA can hamper the TOPO reaction. A 1:1 molar ratio of vector to insert (or about 2-10ng of insert) is recommended.

Can I use the pCR-Blunt or pCR-Blunt-TOPO vector to clone PCR products amplified with Taq DNA polymerase?

No you cannot use pCR-Blunt or pCR-Blunt-TOPO vector to clone PCR products amplified with Taq DNA polymerase. The pCR-Blunt vector is prepared with blunt ends to accept blunt-ended fragments. Due to the terminal transferase activity of Taq DNA polymerase, PCR products amplified with this enzyme have 3'-A overhangs. In order to clone these products into pCR-Blunt, you would need to polish the ends to make them blunt (which usually is not an efficient process). Our TA Cloning Kits or TOPO TA Cloning kits are a better choice for cloning Taq-generated PCR products. TA Cloning kits include a linearized vector with 3'-T overhangs for efficient ligation of Taq-generated PCR products without additional manipulation.

What are the melting temperatures for the M13 Forward (-20) and M13 Reverse primers in the TOPO Cloning and Zero Blunt Kits?

Assuming that the primer is at a 50 nM final concentration and 50 mM final salt concentration, the melting temperatures are: M13 Forward (-20) Primer = 52.7 and the M13 Reverse Primer = 45.3. For use in the control PCR reaction we recommend using an annealing temperature of 56C.

Can TOPO TA cloning vectors and Zero Blunt cloning vectors be used for in vitro transcription to generate probes? Can they be used to do in vitro translation?

Yes, the TOPO TA cloning and Zero Blunt cloning vectors can be used for in vitro transcription. The T7, SP6, and T3 promoters contained in these vectors are active phage promoters, and the corresponding polymerases will transcribe RNA from these promoters.
These vectors are not generally recommended for in vitro translation because most of these vectors contain one or more translational initiation signals (ATG) downstream (3’) of the promoters contained in them, within the multiple cloning sites, which may interfere with attempts to translate an open reading frame in the insert.

Which of the following products will usually give the best cloning efficiency in terms of positive clones and number of colonies: Zero Blunt PCR, Zero Blunt PCR-TOPO, or Zero Blunt PCR-TOPO for Sequencing?

Typically 200-300 colonies are obtained for the TOPO kits with 100% inserts (20/20). For the Zero Blunt kit that uses standard ligase, typically 600-1000 colonies are obtained with 95-100% inserts.

The tube containing the TOPO TA vector arrived yellow instead of the usual red color. What does this indicate? Will this color change affect the activity of the Topoisomerase?

Phenol red is added to the TOPO vector (pCRII, pCR2.1, and pCR4), mostly to make it more visible in the tube. The color of the solution is generally pink at room temperature, but at a low pH phenol red will turn yellow. We have tested acidic conditions (low pH) for the TOPO ligation reaction and found that the activity is not affected. At a pH 2.0, there was no observed decrease in efficiency. However, if the TOPO vector turns dark red, or is blue after adding the PCR product, then the PCR product buffer is too basic (pH 9). High pH does have a negative effect on the TOPO vectors, and the number of resulting colonies in this case will drop.

Note: The Directional TOPO and Zero Blunt TOPO vectors contain bromophenol blue dye rather than phenol red, which results in a blue-colored solution. Consult the product manual to confirm which dye is added to your vector, or contact Technical Support for more information.

What happens when a vector adapted with the Topoisomerase enzyme is stored at -80°C?

-80°C storage is fine. The TOPO vector can be freeze-thawed several times without loss of activity, and the vector is stable for storage at either -20°C or -80°C. However, note that the vector rapidly ages at room temperature (~15 min), which will result in a lower cloning efficiency (a reduction in the number of colonies following transformation). So during active use it should be kept on ice to prevent degradation, and should be quickly returned to the freezer.

Why is there a TOPO Cloning kit "Stop Solution" mentioned in my manual, but it is not supplied in the TOPO Cloning kit?

Stop Solution is a reagent included in older versions of all TOPO kits, and still offered in the TOPO XL kits. It is very similar to the current TOPO cloning kit Salt Solution. Instruction manual versions preceding Version J recommended adding the Stop Solution at the end of the 5 minute TOPO reaction. The basic function of the Stop/Salt Solution is to keep the Topoisomerase from re-binding to the plasmid DNA and nicking the DNA. Later studies found that it was actually more effective to add this component during the TOPO cloning reaction versus afterward, and thus the Stop Solution was replaced with the similar but higher-concentration Salt Solution in most kits.

Is it possible to generate nested deletions using the TOPO Cloning Kit for Sequencing and the ZeroBlunt TOPO PCR Cloning Kits?

Yes. Both vectors (pCR4 and pCR-Blunt II) are designed to allow the creation of nested deletions for sequencing large inserts using the same sequencing primer. In addition, a comprehensive protocol is provided in the TOPO manual for these kits.

How does the pCR4-TOPO and pCR4Blunt-TOPO streamline sequencing?

Both the pCR4-TOPO and pCR4Blunt-TOPO vectors contain a minimized multiple cloning site. There are only 33 base pairs from the sequencing primer sites to the cloning site. This allows for more sequencing read of your insert and less of the vector which an save time analyzing the sequence data.

For Research Use Only. Not for use in diagnostic procedures.