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

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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.

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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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84f74ce4511e0c9531af1182fb636f0f_FAQ

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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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.

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b0ab42fcb7133122b38521d13da7120b_FAQ

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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Why is there a variety of color and size in the colonies from my plated transformation reaction? How should I optimize screening to have the best chance of finding my clone?

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On a typical cloning plate with blue/white screening, there are a few white colonies which do not contain insert. These colonies 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). For the best chance to find a colony with a proper insert, pick clones of a variety of color and pattern for analysis. An insert may generate distinct patterns according to its orientation and size. Even dark blue colonies can have inserts present, if the insert is small and a resulting ORF is in frame with the LacZalpha.

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331316d4efb44682092a006307b9ae3a_FAQ

What polymerases can be used to make PCR products with 3'-A overhangs that are compatible with Invitrogen™ TA Cloning® vectors?

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Here is a list of some commonly known PCR enzymes and indication of their compatibility with TA Cloning® vectors.

1) Enzymes that leave abundant 3'-A overhangs, directly compatible with TA Cloning®:
- Invitrogen™ Taq Polymerase, Platinum® Taq Polymerase, Platinum® PCR Super Mix
- Applied Biosystems® AmpliTaq®, AmpliTaq Gold®, and AmpliTaq® 360 DNA Polymerase
- Ambion SuperTaq™ and SuperTaq™ Plus
- Roche Taq Polymerase and Tth Polymerase
- TaKaRa Taq
- Agilent Taq2000 polymerase, Exo-Pfu Polymerase

2) Enzymes that leave partial A overhangs, generally contain mix of Taq and proofreader and may have lower efficiency with TA Cloning®:
- Invitrogen™ Platinum® Taq High Fidelity and Platinum® PCR Supermix High Fidelity
- Roche Expand System
- TaKaRa LA Taq and Ex Taq

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59a3adea76fadcb6dd9e54c96fc155d1_FAQ

Can a phosphorylated PCR product be TA-cloned?

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Phosphorylated products can be TA-cloned, but not TOPO®-cloned. This is because the necessary phosphate group is already contained within the Topoisomerase-DNA intermediate complex on the TOPO® vectors. The TOPO® plasmid has a 3' phosphate, to which Topoisomerase is covalently bound. The PCR product must provide 5'OH and 3'OH groups to react with the Topoisomerase, and presence of an additional phosphate group will inhibit the Topoisomerase-mediated ligation.

The non-TOPO® TA Cloning® vectors have a 3'OH and a 5' phosphate, and because they involve use of standard ligase, they are compatible with PCR products both with or without 5' and 3' phosphate groups.

Pre-phosphorylated PCR products can be phosphatase-treated (CIP/BIP) to remove the phosphate groups before TOPO® cloning - however, cloning efficiency may be decreased compared with an insert that was never phosphorylated at all.

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98bd65207ee83bfd17ebb0db971eddf9_FAQ

How does the pCR®4-TOPO® and pCR®4Blunt-TOPO® streamline sequencing?

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Both the pCR®4-TOPO® and pCR®4Blunt-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.

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a4fa7175d4757e45eac71a8487751f63_FAQ

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

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Yes. Both vectors (pCR®4 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.

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2de7cf2043693db2ee898479a6e44529_FAQ

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

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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.

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