Chelex

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Extraction

Learning Objectives

• Competence in extraction of different biological stains.

• Knowledge of the theory of DNA Isolation using Chelex ® Extraction methods • Knowledge of advantages and disadvantages of Chelex ® • Overview of 5% Chelex ® extraction procedures

Overview of DNA Process

DNA EXTRACTION DNA QUANTITATION DNA AMPLIFICATION CAPILLARY ELECTROPHORESIS

DNA Facts

• A WBC or epithelial cell (diploid cell) contains approximately 6 pg of DNA – Blood ~1.5  g/drop – Saliva ~50-500 ng/drop – Hair (plucked) ~ 300 ng • Sperm cells (haploid cell) contains approximately 3 pg of DNA – Semen ~ 10  g/drop

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Clean Techniques and Other Considerations

Wear gloves Use a barrier to open tubes (tube opener or Kimwipe) Use only sterile filtered pipette tips Use sterile deionized water Run SOP specified controls (reagent blank, etc) Make Chelex ® per laboratory SOPs

Chelex

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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 and bind metal ions such as magnesium (Mg 2+ ).

• By removing the Mg 2+ from the reaction, nucleases are inactivated and the DNA is protected.

Preparing for Extraction

• Things you may need to prepare in advance: – sterile 1.5 ml microcentrifuge tubes – sterile deionized (DI) water – sterile TE Buffer – sterile spin baskets or separator cups – set incubator to 56ºC – set water bath to 100ºC

Preparing a 5% Chelex

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Suspension

• Add 0.5 g Chelex ® 100 (100-200 mesh, sodium form) to a sterile beaker or suitable glass container • Add10 ml sterile DI water • Mix in a container on a hotplate with a stir bar inside. • Check pH. – Values should be approximately 9 to 11. Adjust pH with a solution of NaOH as needed. – Make fresh daily. Keep mixing on a stir plate while pipetting with a wide bore pipette tip.

Chelex

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Extraction Process

• Many protocols include an initial wash step. Generally this is done by adding ~ 1 ml of sterile DI water to the sample. – This aids in the removal heme and other proteins that inhibit PCR. – This can be done at room temperature or 37ºC. – The incubation times may vary. – After the incubation the sample is centrifuged and the supernatant is removed and discarded. NOTE: leave approximately 30-50  l of supernatant. • A 5 % Chelex ® suspension is added to the tube and incubated at ~ 56 o C – Incubation times can vary from 30 minutes to overnight depending upon the sample type – This step is used to lyse cells. – Chelex ® binds the Mg 2 + ions which inactivates nucleases.

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Extraction Process, cont.

• Some procedures call for the addition of Proteinase K at this step.

• Proteinase K functions to break down proteins, specifically histones which are responsible for packaging DNA.

• The sample is vortexed and then boiled at 100 o C for ~ 8 minutes. • This causes cell membranes to rupture, destroys cellular proteins, and denatures the DNA to yield single-stranded (SS) DNA.

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Extraction Process, cont.

• After the boiling step, some procedures include a vortexing step.

• The tube containing the sample and Chelex ® suspension is then centrifuged, the beads and cellular debris are pelleted, leaving the supernatant containing DNA that is used for quantitation and PCR.

Overview of a Chelex

®

Extraction

~1ml sterile deionized water sample Vortex Incubate 15 - 30 min at 37°C Centrifuge Remove supernatant (except 50-30  l) ~100-200  l Chelex ® 56°C for 30 min to overnight Vortex 100°C 8 minutes Vortex Centrifuge Store Microcon (if needed)

Centrifugal Filter Units

• Many laboratory procedures include a purification and concentration step using a centrifugal filter unit such as the Microcon ® 100 or Centricon ® – Excellent recovery of DNA samples with recoveries typically > 95%. – Used to concentrate, desalt, and purify proteins, antibodies and nucleic acids – Ideal for low level/dilute DNA solutions – Allows products less than 100,000 Daltons to pass through, therefore retaining DNA in the filter

Purification and Concentration

– Initial step • Assemble unit by inserting filter into filtrate/collection tube.

• Add sample and buffer. Centrifuge at low speed.

• Remove the filter unit from the filtrate tube and discard the filtrate. – Wash step • Re-Insert the filter unit into the filtrate tube and add buffer to the sample reservoir and cap. • Centrifuge at low speed and discard the filtrate and filtrate tube.

– Recovery Step • Add the appropriate amount of buffer to the filter.

• Invert and transfer the filter into a new retentate tube.

• Centrifuge at low speed.

• DNA is in the retentate tube.

Storage of DNA

• DNA can be stored refrigerated (4 o C) for short term storage.

• DNA should be stored frozen (approximately -20 o C ) for long term storage.

• Avoid repetitive freeze thawing of DNA, since this can cause degradation.

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Extraction Considerations

• The Chelex ® extraction process denatures double stranded DNA and yields single stranded DNA.

• Care should be taken not to transfer any Chelex ® beads to PCR reaction as this can inhibit PCR • Some studies show low extraction efficiency on low level and compromised samples--organic or other suitable extraction methods may be a better choice for some sample types

Chelex® Extraction-Advantages & Disadvantages

• Advantages: – Quick – Single tube reaction – Non-toxic – Cost effective • Disadvantages: – Ineffective in removing some inhibitors – Yields single stranded DNA (may be problematic with some downstream methods) – May have low yield for compromised and/or low level samples