What is the CloneChecker™ System?

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The CloneChecker™ System is a rapid method for screening recombinant bacterial colonies or liquid cultures of colonies for the presence of target plasmid DNA. The CloneChecker™ System:

- Screens bacterial colonies for plasmid DNA and insert of interest prior to purification
- Screens for inserts >200 bp in plasmids up to 15 kb by size comparison to the original vector or a DNA size marker
- Goes from plated colonies through lysis in less than 5 minutes
- Allows for supercoiled, restriction digest, or PCR analysis
- Works with single-copy plasmids starting with a 2-mm bacterial colony
- Includes sufficient reagents for processing 100 bacterial samples by supercoiled analysis (direct size comparison); and 100 samples by restriction endonuclease or PCR analysis.

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How does the CloneChecker™ system compare to colony PCR?

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The supercoiled analysis method offered by the CloneChecker™ system is an alternative to PCR for screening recombinant bacterial colonies or liquid cultures. Supercoiled analysis is significantly faster and less expensive than colony PCR for screening large numbers of clones. When the cloning efficiency is low, the savings in labor, time and reagent expense using the CloneChecker™ system can be significant. In standard PCR, reaction efficiency drops dramatically for amplicons greater than 1 kb and insert orientation is not usually determined. These limitations are overcome using the CloneChecker™ system.

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1. Can  the TOPO TA cloning kits (TOPO TA for sequencing, Zero blunt, Zero Blunt TOPO, Original TA cloning kit, Dual Promoter cloning kit) be used for in vitro transcription to generate probes? 2. Can they be used to do in vitro translation?

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1. 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.

2. No, 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.

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

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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.

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How does TA Cloning® work?

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Taq polymerase has a non-template-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in our TA Cloning® kits have single, overhanging 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.

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Can I use a DNA polymerase mixture containing both Taq polymerase and a proofreading polymerase for TA Cloning®?

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If you wish to use a polymerase mixture containing Taq polymerase and a proofreading polymerase, Taq must be used in excess with a 10:1 ratio of Taq to the proofreading enzyme to ensure the presence of 3´ A-overhangs on the PCR product. If you use polymerase mixtures that do not have enough Taq polymerase or a proofreading polymerase only, you can add 3' A-overhangs following PCR. See the vector product manuals for details.

Some examples of Taq blends that are compatible with TOPO® TA Cloning® are Platinum® Taq DNA Polymerase High Fidelity and AccuPrime™ Taq DNA Polymerase High Fidelity.

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What are the insert size limitations of TOPO® cloning kits?

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Regular TOPO® TA Cloning® kits are efficient for cloning PCR products up to approximately 2-3kb. With PCR products larger than 3kb, the efficiency of cloning drops significantly. The TOPO® XL PCR Cloning kit has been optimized for TOPO® cloning of long (3-10kb) PCR products.

If using the regular TOPO® kits, here are some tips to improve efficiency:
1. Use crystal violet instead of ethidium bromide (EtBr) to visualize the PCR for gel isolation to avoid DNA nicks
2. Increase incubation time of the TOPO® reaction to 30 mins
3. Keep insert:vector molar ratio low, optimally 1:1
4. Dilute reaction to 20ul, while maintaining same amount of vector and insert. Increase the volume of the salt solution to 3.7ul to compensate for the increase in volume. Diluting the reaction reduces the competition for the vector ends.

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

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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 Life Technologies 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.

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How can I tell whether or not a reagent bottle is pressurizing correctly when using the Procise™ System?

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You can backflush the bottle's pickup line in manual control (there is a specific function for each bottle position on the Procise™ System) and observe its bubbling, which should slow and then stop within a short period (depending upon how full the bottle is) as it pressurizes with argon. If it continues to bubble, either the cap assembly is leaking or the pressurizing or venting valves for the bottle are.

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Why is the conductivity so high in my peptide synthesis (monitored during deprotection)?

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The meter detects any ionic species. A common cause of higher than expected values is a leak of a small amount of resin from the RV into the lines and up to the in-line filters. The use of old or poor quality piperidine or NMP may also give a high background. Standard conductivity measured in micro Siemens/cm is much higher than the sensitivity of this cell. A very small amount of ionic material caused a large change in the reading. Occasionally, Fmoc amino acids have ionic contaminants which give high readings. In-line filters may also be contaminated.

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Are IPTG and X-gal necessary for blue/white screening of colonies?

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With a vector containing the lac promoter and the LacZ alpha fragment (for alpha-complementation), blue/white screening can be used as a tool to select for presence of an insert. X-gal is added to the plate as a substrate for the LacZ-encoded beta-galactosidase enzyme and must always be present to see the blue color in the blue/white screening.

In cases where the lacIq repressor is present (either provided by the host cells on an F' episome, i.e. TOP10F', or expressed from the plasmid), it will repress expression of LacZ from the lac promoter and prevent the formation of the blue color with X-gal. This repression can be reversed by adding IPTG to the media in addition to X-gal, which will inhibit the action of lacIq and re-activate expression from the lac promoter.

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What are some of the competent cell strains offered with your TA Cloning® and TOPO® TA Cloning® vectors, and how should I choose?

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TOP10, TOP10F', DH5a™ and INValphaF' are some of the strains offered with the Original TA Cloning® Kits and the TOPO® TA Cloning Kits. E. coli strains DH5a™,TOP10, and INValphaF' do not have the lacIq repressor and permit constitutive expression from the lac promoter. With these cells, there is no need to add IPTG (inducer) for blue/white screening with X-gal. In contrast, E. coli strain TOP10F' carries the lacIq repressor and requires IPTG inducer to be added to enable blue/white screening. Please note, the F' episome in the INValphaF' strain is different from other strains in that it does not contain the lacIq repressor. Usually, presence of the F' and lacIq is only an advantage if you work with an insert that is potentially toxic to the host cell. If your insert is (potentially) toxic, we recommend using the TOP10F' cells without adding any IPTG. The lacIq repressor will repress expression from the lac promoter and you won't get blue-white screening, but you will still get colonies. This way you can clone a toxic construct.

TOPO® TA cloning® kits are also offered with Mach1™ T1r competent cells. The Mach1™ T1 Phage-Resistant (T1R) E. coli strain is the fastest growing chemically competent strain currently available. Doubling time is approximately 50 minutes, compared with an excess of 74 minutes for other cloning strains. Mach1™ colonies are clearly visible within eight hours of plating the transformation mix, enabling you to plate and pick colonies in the same day. With these cells, there is no need to add IPTG (inducer) for blue/white screening.

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Are the TA or TOPO® TA Cloning® vectors available to purchase without competent cells? What about your Zero Background™, Zero Blunt®, or Zero Blunt® TOPO® Cloning Kits

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Most of our cloning vectors are offered in a complete format, which includes competent cells. While in most cases other cells can be used with our vectors, we cannot guarantee the results you will get with our cloning vectors if you use your own competent cells. For this reason, most TOPO® TA Cloning® vectors can only purchased in a complete kit with cells, although there are a few exceptions. Non-TOPO® vectors are generally available in multiple formats, with and without cells. To get the most current information on available products, visit the Cloning section of our website under Products & Service.

Zero Background™ and Zero Blunt® vectors are available without competent cells provided, but you should especially careful in choosing competent cells to use with them. These vectors contain the ccdB gene for efficient negative selection of clones without insert, and some E. coli strains are not compatible with the mechanism of negative selection by the lethal activity of the ccdB gene product. In particular, cells with the F' episome have a ccd locus containing the ccdA gene, which prevents ccdB protein cell-killing. Therefore, cells without the F' episome are recommended so that only the CcdB protein will be expressed, and its cell-killing ability will not be inhibited or reduced by CcdA.

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Do PCR products produced with Stoffel fragment have sufficient A-overhangs to work with TA Cloning® kit?

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Yes. Stoffel fragment is a 61 kDa modified form of AmpliTaq® DNA Polymerase that lacks 5' to 3' exonuclease activity. It is more thermostable than AmpliTaq® DNA Polymerase, giving it improved performance at the higher denaturating temperatures used with templates that have secondary structure. It more active over a wider range of magnesium concentrations (2 mM to 10 mM MgCl2) than Taq polymerase.

Answer Id: 3332

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Can I use a proofreading enzyme such as Platinum®, Pfx™, Vent, or Pfu with TOPO® TA Cloning® kit?

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Proofreading enzymes possess 3'-5' exonuclease activity (Gene 112:29 (1992)) which removes 3'-A overhangs necessary for TA and TOPO® TA Cloning® kit. Since the PCR products are mostly blunt-ended, the use of these PCR products in TA cloning yields very low cloning efficiencies. We have developed a simple protocol for adding the 3' A overhang to these PCR products so that they can be used in the TA cloning reaction.

Before starting, you will need the following items:
-Taq polymerase
-A heat block equilibrated to 72 degrees C
-Phenol-chloroform
-3M sodium acetate
-100% ethanol
-80% ethanol
-TE buffer

Procedure
-After amplification with Vent, Pfx, or other proofreading polymerases, place samples on ice and add 0.7-1 unit of Taq polymerase per tube. Mix well. It is not necessary to change the buffer or remove the proofreading polymerase. A sufficient number of PCR products will retain the 3'-A overhangs.
-Incubate at 72 degrees C for 8-10 min (do not cycle).
-Place on ice and use immediately in the cloning reaction.
-If you need to store the samples overnight, extract the sample immediately with an equal volume of phenol:chloroform. Extraction with phenol-chloroform removes all of the polymerase. Precipitate the DNA by adding 1/10 volume of 3M sodium acetate and 2X volume of 100% ethanol. Keep at -20 degrees C for 20 min or -80 degrees C for 10-15 min. Centrifuge at maximum speed for 5 min at room temperature to pellet the DNA. Remove the ethanol, rinse the pellet with 80% ethanol, and allow to air dry. Resuspend the pellet in TE buffer to the starting volume of the PCR amplification reaction. The PCR amplification product is now ready for ligation into the TA cloning or TOPO® TA Cloning® vector.

Note: if your amplification has produced more than one PCR product, you may wish to gel-purify the correct fragment after amplification with Pfu or Vent. After purification, add Taq polymerase buffer, dATP, and 0.5 unit of Taq polymerase and incubate 10-15 min at 72 degrees C. Proceed directly to the cloning reaction.

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