What does Chelex do in PCR?

What does Chelex do in PCR?

Chelex protects the sample from DNases that might remain active after the boiling and could subsequently degrade the DNA, rendering it unsuitable for PCR. After boiling, the Chelex-DNA preparation is stable and can be stored at 4°C for 3–4 months.

How do Chelex beads work?

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.

How do you use Chelex resin?

1. Chelex resins may be used with either a batch method or a column method.
2. The batch method is the addition of resin directly into the sample followed by stirring.
3. The column method involves pouring a column with the Chelex resin and passing the sample through to achieve the separation.
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What is the purpose of Chelex beads used in buccal cell DNA 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.

Do Chelex beads inhibit PCR?

Chelex can chelate a large amount of divalent ions that may be donated by the sample, and the Chelex beads can be easily removed so that they will not interfere with subsequent PCR amplifications that require Mg++.

What is Chelex method?

The Chelex method has been used with amplification and typing at the HLA DQ alpha locus to obtain the DQ alpha genotypes of many different types of samples, including whole blood, bloodstains, seminal stains, buccal swabs, hair and post-coital samples.

Why is Chelex a PCR inhibitor?

Chelex 100 Resin is designed to strongly bind divalent metal ions such as Mg2+ that are required for the activity of metallo-nucleases. The addition of Chelex 100 effectively inhibits nuclease activity in complex samples and stabilizes samples for downstream PCR applications.

How much Chelex do I add?

10% (wt/vol) Chelex 100: Add 1 g Chelex 100 resin to water (MilliQ or NANOpure) and bring up to a final volume of 10 ml. Store at room temperature, 20–25 °C.

Is Chelex a lysis buffer?

Chelex is a chelating ion-exchange resin that binds polar components of cells leading to cell lysis. The remaining non-polar DNA remains in the aqueous solution above the Chelex. This resin prevents DNA degradation by binding (chelating) metal ions (Mg2+) that catalyze the breakdown of DNA.

Why do we add ammonium acetate in DNA extraction?

The presence of debris in the DNA solution may result in degradation of DNA on long term storage and inhibition of the polymerase chain reaction. We used ammonium acetate to precipitate proteins and a sodium acetate- isopropanol mixture to pellet out DNA which was washed with ethanol.

How do you make a 5% Chelex?

Chelex-based extraction procedures Make 20% and 5% (wt/vol) stock solutions in sterile, distilled water to a final volume of 100-300 ml.

What is Chelex extraction used for?

The Chelex method has been used with amplification and typing at the HLA DQα locus to obtain the DQα genotypes of many different types of samples, including whole blood, bloodstains, seminal stains, buccal swabs, hair and post-coital samples.

What is Chelex made of?

•Chelex is composed of styrene divinylbenzene copolymers containing paired iminodiacetate ions that act as chelating groups in binding polyvalent metal ions such as magnesium (Mg2+). •By removing the Mg2+ from the reaction, nucleases are inactivated and the DNA is protected.

What is Chelex extraction?

Dr.L.Yatawara Chelex Extraction •Chelex 100 is an ion exchange resin that is added as a 5% solution (wt/vol). •Chelex is composed of styrene divinylbenzene copolymers containing paired iminodiacetate ions that act as chelating groups in binding polyvalent metal ions such as magnesium (Mg2+).

How does chelex 100 differ from other cation exchange resin?

The carboxylic acid groups of Chelex 100 resin classify it as a weak cation exchange resin, but it differs from other exchangers in this class by featuring uniquely high selectivities for metal ions and much higher bond strengths.

How do you use Chelex in blood staining?

•A 5% solution of Chelex is added to a blood stain or liquid blood and incubated at 56C for 30 minutes. This step is used to lyse red cells and remove contaminants and inhibitors such as heme and other proteins. •The sample is then heated at 100C for 8 minutes.