What polymerase is used for TOPO cloning?
This kit is specifically designed to clone blunt-end PCR products generated by thermostable proofreading polymerases such as Invitrogen™ Platinum™ SuperFi™ DNA Polymerase.
What does TOPO cloning do?
TOPO cloning is a molecular biology technique in which DNA fragments are cloned into specific vectors without the requirement for DNA ligases. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3′-end of the PCR products.
What enzyme is specifically associated with the TOPO cloning kit and how does it work?
The key to TOPO cloning is the enzyme DNA topoisomerase I, which functions both as a restriction enzyme and as a ligase. Its biological role is to cleave and rejoin DNA during replication.
Does PCR product have 5 phosphate?
Alternatively, primers for PCR can be ordered with 5′ phosphate to avoid the need to separately phosphorylate the PCR product with a kinase.
Is DNA ligase used in PCR?
The equivalent of DNA polymerase I and DNA ligase are also unnecessary due to the absence of RNA primers and Okazaki fragments during the process of PCR. Since PCR requires very high temperatures as you will see, a typical DNA polymerase cannot be used since it will be denatured by the intense heat.
How do you design primers for TOPO cloning?
If required, SnapGene will automatically design appropriate insert-specific primers for directional TOPO® cloning.
- Open the TOPO® TA Cloning Dialog.
- Choose the Insert.
- Choose the Vector.
- Design Primers for the Insert.
- Confirm the Predicted Insert Sequence and Fusion Product.
- Rename the New Primers.
Is TOPO cloning directional?
This occurs at both ends of the linearized vector, creating a circular vector+insert. In practise TOPO-cloning is not directional, so the insert is equally likely to ligate in both directions.
How does PCR Blunt II Topo work?
pCR™Blunt II-TOPO® clone selection pCR™-Blunt II-TOPO® allows for direct selection of recombinants via disruption of the lethal E. coli gene, ccd B. The vector contains the ccd B gene fused to the C-terminus of the LacZα fragment.
What cells are used in Zero Blunt® Topo® PCR cloning?
The Zero Blunt® TOPO® PCR Cloning Kit for direct insertion of blunt-ended PCR products into a plasmid vector can be purchased with a variety of competent cells that deliver different advantages depending upon your needs: General cloning: TOP10 cells (Cat.
What primers are included in the PCR™4blunt Topo® kits?
The pCR™4Blunt TOPO® vector has sites for 4 common sequencing primers: M13 forward, M13 reverse, T7, and T3. The kits include an aliquot of each.
How many preps are in a Zero Blunt Topo® PCR kit?
Selected Zero Blunt® TOPO® PCR Cloning Kits are available in combo kits combined with a PureLink™ Quick Plasmid Miniprep Kit (50 preps) for fast plasmid purification of your TOPO®-cloned inserts for downstream analysis.