- 1 How does NEBuilder work?
- 2 What is a HiFi reaction?
- 3 How does Neb HiFi assembly work?
- 4 What is infusion cloning?
- 5 What is the purpose of colony PCR?
- 6 How do you do a Gibson Assembly?
- 7 How does Golden Gate assembly work?
- 8 How do you do site directed mutagenesis?
- 9 What is LR recombination?
- 10 What is annealing temp?
- 11 How do you design a primer?
How does NEBuilder work?
NEBuilder HiFi DNA Assembly enables virtually error-free joining of DNA fragments, even those with 5´- and 3´-end mismatches. This flexible kit enables simple and fast seamless cloning utilizing a proprietary high-fidelity polymerase. Find out why NEBuilder HiFi is the next generation of DNA assembly and cloning.
What is a HiFi reaction?
GeneArt Gibson Assembly HiFi Master Mix enables DNA assembly and cloning via a technique that allows multiple overlapping DNA fragments to be seamlessly linked in a one-step, 15–60 minute isothermal reaction. DNA fragments of different lengths are uniformly assembled using complementary overlaps between fragments.
How does Neb HiFi assembly work?
NEBuilder HiFi DNA Assembly Reaction Protocol NEB recommends a total of 0.03–0.2 pmol of DNA fragments when 1 or 2 fragments are being assembled into a vector, and 0.2–0.5 pmol of DNA fragments when 4 –6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases.
What is infusion cloning?
Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods.
What is the purpose of colony PCR?
Colony PCR is a method for rapidly screening colonies of yeast or bacteria that have grown up on selective media following a transformation step, to verify that the desired genetic construct is present, or to amplify a portion of the construct.
How do you do a Gibson Assembly?
- Design your plasmid and order primers (see figure to the right).
- Generate DNA segments by PCR.
- Run PCR product on an agarose gel to check for size and yield.
- Combine segments in Gibson Assembly Reaction.
- Transform the DNA into bacteria and screen for the correct plasmid product by Restriction Digest.
How does Golden Gate assembly work?
Golden Gate cloning or Golden Gate assembly is a molecular cloning method that allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIs restriction enzymes and T4 DNA ligase. This assembly is performed in vitro.
How do you do site directed mutagenesis?
In this method, a fragment of DNA is synthesized, and then inserted into a plasmid. It involves the cleavage by a restriction enzyme at a site in the plasmid and subsequent ligation of a pair of complementary oligonucleotides containing the mutation in the gene of interest to the plasmid.
What is LR recombination?
The LR Reaction, again is a recombination reaction between attL and attR sites. The reaction generates an expression clone and is catalyzed by recombinant proteins. Recombination between these sites generates two molecules. One molecule contains the DNA segment of interest, the other molecule is a by-product.
What is annealing temp?
The annealing temperature is the temperature used in the annealing step of a PCR reaction, which is highly dependent on the Tm of primers. Thereby, the annealing temperature is usually set as a few degrees (3-6) lower than the lowest Tm of the primers.
How do you design a primer?
PCR Primer Design Tips
- Aim for the GC content to be between 40 and 60% with the 3′ of a primer ending in G or C to promote binding.
- A good length for PCR primers is generally around 18-30 bases.
- Try to make the melting temperature (Tm) of the primers between 65°C and 75°C, and within 5°C of each other.