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title: "TheNessLab : DNA Extraction: Chelex Method"

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TheNessLab : DNA Extraction: Chelex Method

Created by Wen Chen on May 11, 2018

Use this protocol to create DNA stocks for PCR reactions. 

Materials:

• 5% w/v Chelex-100: i.e. 0.5g Chelex-100 + 10 mL autoclaved HPLC water
• at least 1mm diameter cell culture on agar or 10 uL cell culture
• Incubator, centrifuge, vortexor
• Ice in styrofoam box
• Inoculating loops (only if using cell culture on agar)

Procedure:

Before you start, turn on the incubator for preheat and prepare ice in a styrofoam box.

1. In a sterilized tube, add one 1mm-diameter of cell culture using inoculating loop or 10 uL cell culture in 50 uL 5% Chelex-100.
2. Vortex for 5 seconds.
3. Spin at 10,000g (rcf) at 20C for 1 minute.
4. Incubate at 100C for 10 minutes.
5. Cool down on ice for 1 minute.
6. Repeat 2 and 3. 
7. Use 1 uL of supernatant for 20 uL PCR reaction or store extracted supernatant in -20C freezer.
   • Make sure extracted supernatant does not contain Chelex. Chelex will interfere with PCR. 
   • Use Qubit to quantify DNA concentration if necessary.

Troubleshooting:

• If DNA concentration is too low for PCR when using liquid culture, try transfering the whole culture (<2mL volume) into a 2 mL and spin the culture at 10,000g. Resuspend the pellet in 50 uL 5% Chelex-100 and continue from Step 2.
• If DNA concentration is too low for PCR when using agar culture, take a bigger swab with inoculating loop.

Related articles

Modified from: Cao, M., Fu, Y., Guo, Y. et al. Protoplasma (2009) 235: 107. https://doi.org/10.1007/s00709-009-0036-9

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