How much PCR product for TOPO cloning?
Table of Contents
How much PCR product for TOPO cloning?
0.5 to 4 µl
Performing the TOPO Cloning Reaction
| Reagent* | Chemically Competent E. coli | Electrocompetent E. coli |
|---|---|---|
| Fresh PCR product | 0.5 to 4 µl | 0.5 to 4 µl |
| Salt Solution | 1 µl | — |
| Dilute Salt Solution (1:4) | — | 1 µl |
| Sterile Water | add to a final volume of 5 µl | add to a final volume of 5 µl |
What is blunt end PCR product?
Blunt-end cloning is a convenient way to clone polymerase chain reaction (PCR) products generated by proof-reading DNA polymerase. However, it is a time consuming procedure to prepare the linearized blunt-end vector, which usually involves plasmid extraction and restriction enzyme digestion.
What is the role of the ccdB gene in the donor and destination vectors?
The ccdB gene is present in the donor vectors and the destination vectors prior to recombination, and it is exchanged with the gene of interest during the BP or LR reactions. Since the CcdB protein inhibits the growth of CcdB sensitive E. coli strains, most colonies should contain the desired, recombined construct.
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.
How do you sequence PCR products?
You must remove all PCR primers and unincorporated nucleotides before the product is sequenced. Sequencing uses only one primer instead of the two used in PCR. If you do not remove both primers, you will get two sequences superimposed on each other that are not readable.
How do you identify a blunt end?
In sticky ends, one strand is longer than the other (typically by at least a few nucleotides), such that the longer strand has bases which are left unpaired. In blunt ends, both strands are of equal length – i.e. they end at the same base position, leaving no unpaired bases on either strand.
Is ccdB toxic to mammalian cells?
The particular property of the ccd system is that it targets the DNA gyrase, a topoisomerase absent from the cells of higher eukaryotes. The poison is therefore not toxic for mammalian cells.20. Positive selection of recombinant DNA by CcdB.
Is TOPO cloning directional?
Directional TOPO cloning enables cloning of blunt-ended PCR products in a 5´→3´ orientation directly into a expression vector using a 5-minute ligation reaction, thereby eliminating subcloning steps and saving you time.
What is TOPO TA Cloning?
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.
