What is sequenase?
Description: Sequenase™ Version 2.0 DNA Polymerase is a genetically engineered form of T7 DNA polymerase. The genetic changes in this subunit (a deletion of 28 amino acids accomplished by in vitro mutagenesis) eliminate all measurable exonuclease activity without changing the DNA polymerase activity.
What is Klenow polymerase enzyme?
Klenow Fragment is the large fragment of DNA Polymerase I that retains its 5’→3′ polymerase, 3’→5′ exonuclease and strand displacement activities. The enzyme lacks the 5’→3′ exonuclease activity of intact DNA polymerase I. Klenow retains the polymerization fidelity of the holoenzyme without degrading 5′ termini.
What is Klenow fragment used for?
The Klenow fragment is extremely useful for research-based tasks such as: Synthesis of double-stranded DNA from single-stranded templates. Filling in receded 3′ ends of DNA fragments to make 5′ overhang blunt. Digesting away protruding 3′ overhangs.
Is a modified T7 polymerase lacking any exonuclease activity?
T7 DNA polymerase lacks the 5′–3′ exonuclease activity found in E. coli DNA polymerase I but does possess a strong 3′–5′ single and double stranded DNA exonuclease activity (Hori et al., 1979b).
Does Klenow leave phosphorylated ends?
It is the same 5′ end with a phosphate that was left behind after restriction digestion. So there is no need to phosphorylate your DNA product. There is no need to phosphorylate after blunting.
What is the Sequenase™ Version 2 0 DNA sequencing kit?
The Sequenase™ Version 2. 0 DNA Sequencing Kit features Sequenase Version 2. 0 DNA Polymerase, the standard for high quality manual DNA sequencing. Sequenase Version 2. 0 DNA polymerase is a genetically engineered form of T7 DNA polymerase which retains extraordinary polymerase activity with virtually no 3’→5′ exonuclease activity.
How is Sequenase different from the wild-type enzyme?
Unlike the wild-type enzyme it has virtually no 3’→5′ exonuclease activity. Sequenase Version 2.0 is highly processive, incorporates nucleotide analogs (dlTP, thio-dNTPs, dideoxy-NTPs, etc.), is not impeded by secondary structures, and can carry out strand displacement synthesis.
How do you store Sequenase enzyme?
Never store Sequenase enzyme in a frost-free freezer since the temperature rises above 0°C daily. If enzyme dilution buffer (no glycerol) is to be used, only dilute the amount of enzyme which is to be used that day. Dilute into ice-cold buffer and keep on ice until use.
What is the role of thiocyanate in dideoxy sequencing?
It is an excellent enzyme for dideoxy-sequencing, and is useful in other applications, especially where the absence of associated exonuclease activity is desirable.