Principle: Chelex resin works by preventing DNA degradation from degradative enzymes (DNases) and from potential contaminants that might inhibit downstream analyses. In general, the Chelex resin will trap such contaminants, leaving DNA in solution. This releases DNA into solution.
- 1 What does Chelex do to the cheek cell extraction?
- 2 What is Chelex and how does it function?
- 3 Why is Chelex essential during cell lysis?
- 4 What is a chelator in DNA extraction?
- 5 Why is a saline solution used for isolating cheek cells?
- 6 What is Chelex made of?
- 7 How do you make a Chelex solution?
- 8 How PCR works step by step?
- 9 How is DNA extracted from dried blood?
- 10 Is Chelex toxic?
- 11 How do you prevent DNA degradation during extraction?
- 12 What is the purpose of the InstaGene Chelex resin used in our PCR sample preparations?
- 13 What is the role of chelating agent in DNA extraction?
- 14 Why chelating agents are used in extraction of metal ion?
- 15 How does elution buffer work in DNA extraction?
What does Chelex do to the cheek cell extraction?
The Chelex beads will bind divalent magnesium ions (Mg++). These ions often serve as cofactors for nucleases that will degrade your DNA sample and may interfere with the enzyme (Taq polymerase) used in the reaction. By removing magnesium ions, the degradation of genomic DNA by nucleases is reduced.
What is Chelex and how does it function?
Chelex 100 is a chelating material from Bio-Rad used to purify other compounds via ion exchange. It is noteworthy for its ability to bind transition metal ions. Chelex protects the sample from DNases that might remain active after the boiling and could subsequently degrade the DNA, rendering it unsuitable for PCR.
Why is Chelex essential during cell lysis?
Chelex is a chelating ion-exchange resin that binds polar components of cells leading to cell lysis. This resin prevents DNA degradation by binding (chelating) metal ions (Mg2+) that catalyze the breakdown of DNA.
What is a chelator in DNA extraction?
A common chelating agent used in molecular biology is Ethylenediaminetetraacetic Acid (EDTA). Metal ions will compete with protons in order to bind with EDTA. These metal ions are responsible for the activity of DNAse, an enzyme that breaks the phosphodiester bonds of DNA, thus cleaving it.
Why is a saline solution used for isolating cheek cells?
Why the salt water? In this protocol, the aim is to get a sample of DNA from cheek cells. Your saliva, after rinsing your mouth will naturally contain cheek cells, which will be broken open during the protocol to release the DNA. The salt, i.e. sodium chloride, is used to stabilise the DNA, once it has been released.
What is Chelex made of?
Chelex 100 Resin is made of a styrene divinylbenzene copolymer containing paired iminodiacetate ions, which act as chelating groups in binding polyvalent metal ions.
How do you make a Chelex solution?
Dissolve 9 g NaCl (mw 58.44) in 700 ml deionized or distilled water in clean container. Add water to bring total solution volume to 1000 ml. 10% Chelex
- Weigh out 1 g of Chelex 100 (100-200 mesh, sodium form from BioRad).
- Add 50 mM Tris to dry Chelex to make 10 ml of solution.
- Adjust pH to 11 using 4 N NaOH.
How PCR works step by step?
Amplification is achieved by a series of three steps: (1) denaturation, in which double-stranded DNA templates are heated to separate the strands; (2) annealing, in which short DNA molecules called primers bind to flanking regions of the target DNA; and (3) extension, in which DNA polymerase extends the 3′ end of each
How is DNA extracted from dried blood?
Blood is, therefore, the preferred source of DNA samples. They can be collected more efficiently in the field and transported and stored for later DNA extraction without the need for refrigeration. DNA has traditionally been extracted from dried blood using dried blood spots on filter paper.
Is Chelex toxic?
Not a dangerous substance or mixture according to the Globally Harmonised System (GHS). Inhalation May be harmful if inhaled. May cause respiratory tract irritation. Skin May be harmful if absorbed through skin.
How do you prevent DNA degradation during extraction?
In summary, the key steps to prevent DNA degradation are:
- Correct handling & storage of starting material.
- Perform Extractions at 4°C, on ice or in the cold.
- Inhibit nuclease activity.
- Store purified DNA correctly.
What is the purpose of the InstaGene Chelex resin used in our PCR sample preparations?
InstaGene matrix, made with a specially formulated 6% w/v Chelex resin, makes DNA sample preparation fast, easy, and cost-effective, providing PCR-quality template DNA in less than an hour. The Chelex matrix binds to PCR inhibitors rather than DNA, preventing DNA loss due to irreversible DNA binding.
What is the role of chelating agent in DNA extraction?
The EDTA works as a chelating agent in the DNA extraction. It chelates the metal ion present into the enzymes and as we all know that the metal ions are the cofactor which increases the activity of the enzyme. By chelating the metal ions, it deactivates the enzyme, therefore, reduces the activity of DNase and RNase.
Why chelating agents are used in extraction of metal ion?
Chelation therapy is an antidote for poisoning by mercury, arsenic, and lead. Chelating agents convert these metal ions into a chemically and biochemically inert form that can be excreted.
How does elution buffer work in DNA extraction?
Elution. DNA is soluble in low-ionic-strength solution such as TE buffer or nuclease-free water. When such an aqueous buffer is applied to a silica membrane, the DNA is released from the silica, and the eluate is collected.