TA Cloning® Kit, with pCR™2.1 Vector and One Shot® INVαF' Chemically Competent E. coli, no manual - FAQs

When using a proofreading polymerase such as Pfx, ThermalAce, Pfu, or Vent, is it necessary to neutralize or remove the enzyme after regenerating A-overhangs with a Taq polymerase?

It is best practice to remove the enzymes via phenol-chloroform extraction to prevent the proofreading enzyme from removing the A-overhang again after Taq incubation. If not done, TA cloning efficiencies could be 4-10 fold lower. Alternatively, the PCR product can be cleaned up by gel purification or PCR cleanup column.

Can a phosphorylated PCR product be TA-cloned?

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.

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

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
- Invitrogen 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

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

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.

Can you ligate PCR products into a TA vector at room temperature instead of 14°C?

Comparing a 4 hour ligation at 14°C vs. a 2 hour ligation at room temp, approximately two times more colonies will be observed with the 4 hour ligation at 14°C. For room temperature ligations, the TOPO TA Cloning kits will give much higher yields with a reaction time of only 5 minutes.