Dahlquist:DNA Extraction from Human Hair
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Overview
These protocols describe the steps necessary to obtain DNA from human hair for downstream PCR applications.
The first protocol was adapted from:
- Suenaga, E., & Nakamura, H. (2005). Evaluation of three methods for effective extraction of DNA from human hair. Journal of chromatography B, 820(1), 137-141. DOI: 10.1016/j.jchromb.2004.11.028
- See discussion at ResearchGate
The second protocol uses a commercial kit from Qiagen.
Protocol 1
Recipes & Reagents
10% Chelex
Makes 10 mL. Store at room temperature.
- Weigh out 1.0 g of Chelex 100 (100-200 mesh, sodium form).
- Add 50 mM Tris to the dry Chelex, to make 10 mL of solution.
- Adjust the pH to 11, using concentrated NaOH.
- Recipe can be increased to make 50 mL.
Note that the Suenaga et al. article uses a 5% solution and does not specify the buffer or pH of the solution. The 10% solution described here from the lab manual for Biology 478 works well.
Proteinase K
- Proteinase K, Molecular Biology grade, 800 units/mL, ~20 micrograms/mL, catalog #P8107S
Protocol 1
Prepare Hair
- Set heat block to 100 degrees Celsius.
- Pluck 4 hairs from head, making sure follicles are attached.
- Tape down hair end (opposite of follicle end) and trim follicle end of hair to 0.5 cm length with clean scissors.
- Place the trimmed hairs into 0.5 mL microcentrifuge tube.
- Wash with 500 μL of 100% ethanol.
- Add 500 μL of 100% ethanol to the tube.
- Flash spin.
- Pipette off excess ethanol before drying.
- Air dry for 10 minutes in fume hood with cap off until all ethanol has evaporated.
Chelex extraction
- Add 200 μL of 10% Chelex solution to tube with hairs using P1000 tips that had had the tip cut off to make a larger aperture.
- Vortex Chelex solution immediately before pipetting and be sure to pipette from lower down in the tube.
- Add 5 μL of 20 μg/μL Proteinase K (NEB, 100 μg) to microcentrifuge tube with hairs and mix well.
- Incubate in 55°C water bath for 1 hour.
- Vortex and incubate in a heat block set to 100°C for 8 minutes.
- Spin in a microcentrifuge at 10,000 - 15,000 X g for 2-3 minutes.
- Transfer 100 μL of the supernatant to a 1.5 mL microcentrifuge tube.
- Use supernatant immediately for PCR or store at -20°C.
QIAamp DNA Investigator Kit (Protocol 2)
QIAamp DNA Investigator Kit (50) Cat# 56504
Recipes & Reagents
Buffer ATL
- Before starting the procedure, check whether precipitate has formed in Buffer ATL.
- If necessary, dissolve by heating to 70°C with gentle agitation.
Buffer AL
- Before starting the procedure, check whether precipitate has formed in Buffer AL.
- If necessary, dissolve by heating to 70°C with gentle agitation.
Buffer AW1
- Add 25 ml ethanol (96–100%) to the bottle containing 19 ml Buffer AW1 concentrate.
- Tick the check box on the bottle label to indicate that ethanol has been added.
- Reconstituted Buffer AW1 can be stored at room temperature (15–25°C) for up to 1 year.
- Before starting the procedure, mix the reconstituted Buffer AW1 by shaking.
Buffer AW2
- Add 30 ml ethanol (96–100%) to the bottle containing 13 ml Buffer AW2 concentrate.
- Reconstituted Buffer AW2 can be stored at room temperature (15–25°C) for up to 1 year.
- Before starting the procedure, mix the reconstituted Buffer AW2 by shaking.
1 M DTT (dithiothreitol) Stock Solution
- Freeze in aliquots at -20°C.
Buffer ATE with Carrier RNA
- Add 310 µl Buffer ATE to the tube containing 310 µg lyophilized carrier RNA to obtain a solution of 1 µg/µl.
- Dissolve the carrier RNA thoroughly
- Divide it into conveniently sized aliquots
- Store at –30°C to –15°C. Do not freeze–thaw the aliquots of carrier RNA more than 3 times.
Protocol
Before Starting
- Equilibrate Buffer ATE or distilled water for elution to room temperature (15–25°C)
- Calculate the volume of Buffer AL and dissolved carrier RNA needed per batch of samples by multiplying the number of samples to be simultaneously processed by the volume of Buffer AL used (300μL). To allow for pipetting errors, always prepare enough buffer for processing two extra samples.
- Add 1μL of carrier RNA per 300μL of Buffer AL.
- Gently mix Buffer AL and dissolved carrier RNA by inverting the tube 10 times. To avoid foaming do not vortex.
- Buffer AL containing carrier RNA is stable at room tempaerature (15-25°C) for up to 48 hours.
- Set heat block or water bath to 56°C and 70°C
- Thaw aqueous 1 M DTT (dithiothreitol)
- Before starting the procedure, check whether precipitate has formed in Buffer AL or ATL.
- If necessary, dissolve by heating to 70°C with gentle agitation.
Prepare Hair
- Pluck 4 hairs from head, making sure follicles are attached.
- Tape down hair end (opposite of follicle end) and trim follicle end of hair to 0.5 cm length with clean scissors.
Procedure
- Label the tubes you will need (# of samples).
- In each 1.5mL microcentrifuge tube, add 300μL Buffer ATL, 20μL Proteinase K, and 20μL 1M Dithiothreitol (DTT).
- Add prepared hair into respective microcentifuge tube, close tube and mix.
- Mix by vortexing/flash spin for 10 seconds.
- Incubate at 56ºC for at least 1 hour
- If using heat block or water bath, vortex for 10 seconds every 10 minutes
- Spin down for 3-5 seconds
- Add 300μL Buffer AL (with carrier RNA) to microcentrifuge tubes, close tube and mix.
- Mix by vortexing/flash spin for 10 seconds.
- Confirm mixture is homogenous. Mix for longer if not.
- Precipitate may form, this has no effect.
- Incubate at 70ºC for 10 minutes
- If using heat block or water bath, vortex for 10 seconds every 3 minutes
- Spin down for 3-5 seconds
- Add 150μL Ethanol (96-100%) to microcentrifuge tubes, close tube and mix.
- Mix by vortexing/flash spin for 15 seconds.
- Confirm mixture is homogenous. Mix for longer if not.
- Spin down for 3-5 seconds
- Transfer supernatant from microcentrifuge tube to a QIAamp Column in a 2mL Collection Tube and close lid.
- Do not wet rim.
- Spin for 1 minute at 6,000 X g (8000 rpm) in the microcentrifuge.
- If lysate remains, spin again at higher speed
- Place the column in new collection tube. Discard the collection tube with flow-through
- Add 500 μL of Buffer AW1 to the column.
- Do not wet rim.
- Spin for 1 minute at 6,000 X g (8000 rpm) in the microcentrifuge.
- Place the column in new collection tube. Discard the collection tube with flow-through.
- Add 700 μL of Buffer AW2 to the column.
- Do not wet rim.
- Spin for 1 minute at 6,000 X g (8000 rpm) in the microcentrifuge.
- Place the column in new collection tube. Discard the collection tube with flow-through. Make sure that flow-through has not come in contact with the column. If it has, spin again.
- Add 700 μL of ethanol (96-100%) to the column.
- Do not wet rim.
- Spin for 1 minute at 6,000 X g (8000 rpm) in the microcentrifuge.
- Place the column in new collection tube. Discard the collection tube with flow-through.
- Spin at full speed (15,000 X g; 14,000 rpm) for 3 min to dry the membrane completely.
- It is important that this is completely dry!
- Place the column in 1.5 mL microcentrifuge tube with cap cut off. Discard the collection tube with flow-through.
- Open lid of QIAamp MinElute Column, and incubate at room temperature for 10 min or at 56°C for 3 min.
- Apply 50 µl room temperature Buffer ATE or distilled water to the center of the membrane.
- Close the lid and incubate at room temperature for 5 min
- Centrifuge at full speed (20,000 x g; 14,000 rpm) for 1 min.
- Carefully transfer eluate to a labeled 1.5 mL microcentrifuge tube with a lid. Use immediately in PCR or store at -20°C.
