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Protocol
Rootless Hair DNA Purification Protocol
Components
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Shipping conditions: At ambient temperature
Handling And Storage: Store The Kit Components According To The Table Above On Arrival.
Components
BcMag™ HO-DNA Beads
BcMag™ Hair Pigment Removal Beads
1x Lysis Buffer
10x Binding buffer
1x Elution Buffer
Proteinase K
Proteinase K Suspension Buffer
DTT
Storage
4°C
4°C
4°C
4°C
4°C
-20°C
4°C
-20°C
Cat. No. AD101 (50 Preps)
0.5 ml
0.3 ml
5 ml
1.5 ml
0.75 ml
10 mg
0.5 ml
75 mg
Cat. No. AD102 (100 Preps)
1.0 ml
0.6 ml
10 ml
3.0 ml
1.5 ml
20 mg
1.0 ml
150 mg
Shipping conditions: At ambient temperature
Handling and Storage: Store the kit components according to the table Above on arrival.
Introduction
DNA purification is an essential step in the field of molecular biology and genetics. It is the process of isolating DNA from a sample and removing any impurities that may interfere with downstream analysis. Hair is a common source of DNA, and rootless hair, in particular, provides a new opportunity for DNA analysis. BcMag™ Rootless Hair DNA Purification Protocol is a new method for purifying DNA from rootless hair. This protocol is unique because it allows for the efficient and sequential extraction of PCR inhibitor-free nucleic acids from single rootless hair.
Workflow and Results
1.
Lyse the hair at 95°C for 2 hours.
2.
Add magnetic beads to bind the DNA.
3.
Wash the beads.
4.
Elute DNA from the beads.
The BcMag™ Rootless Hair DNA Purification Protocol has several advantages over traditional DNA purification methods. One of the most significant benefits is its ability to extract DNA from a single rootless hair with high efficiency, enabling the analysis of small or limited amounts of DNA. Another advantage is that it produces nucleic acids that are free of PCR inhibitors, which is essential for reliable downstream analysis.
The protocol uses a sequential process to extract nucleic acids from a single rootless hair efficiently. The process begins by lysing the hair to release the DNA into a solution. The DNA is then purified using proprietary magnetic beads, which remove any impurities. Finally, the purified DNA is eluted and ready for downstream analysis.
Producing nucleic acids that are free of PCR inhibitors is essential for accurate and reliable downstream analysis. PCR inhibitors can interfere with the polymerase chain reaction (PCR), which is a commonly used method for amplifying DNA. If PCR inhibitors are present in the DNA sample, they can cause the PCR reaction to fail or produce inaccurate results. By producing PCR inhibitor-free nucleic acids, the BcMag™ Trace Amount of Rootless Hair DNA Purification Protocol ensures reliable and accurate downstream analysis.
PROTOCOL
The following protocol is an example. The protocol can be scaled up or down as needed.
Notes
●
DNA Yield: Varies (depends on sample size and type. Typically, 5cm hair shaft has 100 pg)
●
DNA Size: Varies (depends on the quality of starting material)
●
Quantification of the nucleic acids: Since there is a trace amount of DNA from the sample, use fluorescent dye to quantify. OD260 methods such as Nanodrop are not suitable.
●
For long-term storage, store the extracted nucleic acids at -20°C.
Materials Required by the User
●
95–100% ethanol
●
80% isopropyl alcohol
●
10x Triton X-100 (1% Triton X-100)
●
10x NaCl (5M NaCl)
●
Digital Multi Heat Block
●
Microcentrifuge tubes, 2 ml
●
Aerosol-resistant micropipette tip
●
Magnetic rack: Based on sample volume, the user can choose one of the following magnetic Racks:
BcMag™ Rack-2 for holding two individual 1.5 ml centrifuge tubes (Bioclone, Cat. No. MS-01)
BcMag™ Rack-6 for holding six individual 1.5 ml centrifuge tubes (Bioclone, Cat. No. MS-02)
BcMag™ Rack-24 for holding twenty-four individual 1.5-2.0 ml centrifuge tubes (Bioclone, Cat. No. MS-03)
BcMag™ Rack-50 for holding one 50 ml centrifuge tube, one 15 ml centrifuge tube, and four individual 1.5 ml centrifuge tubes (Bioclone, Cat. No. MS-04)
Sample
Example Sample Input
Rootless Hair
Up to 1mg – 5mg
A. Master Mix Preparation
! IMPORTANT !
●
Proteinase K preparation: Provide protease K as lyophilized powder and dissolve at a 20 mg/ml concentration in Proteinase K Suspension Buffer. For example, 10 mg dissolved in 500µl of Proteinase K Suspension Buffer. Divide the stock solution into small aliquots and store at -20°C. Each aliquot can be thawed and refrozen several times but should then be discarded.
●
DTT solution preparation: Provide DTT as powder and make 20% solution with ultrapure water. For example, 60 mg dissolved in 300µl of ultrapure water. It is stable for years at -20°C. Prepare in small aliquots, thaw it on ice, and use and discard. Store them in the dark (wrapped in aluminum foil) at -20°C. Do not autoclave DTT or solutions containing it. Avoid multiple freeze-thaw cycles.
1.
Preheat the Thermocycler or digital multi-heat block to 95°C.
2.
Prepare a fresh Master Mix following Table for the number of samples to be processed, plus 10% more (e.g., if you have 10 samples, prepare Master Mix for 11). Add the following components to the reservoir.
Components
1 Well (75 μL Reaction Volume)
1x Lysis Buffer
67.5 μL
20% DTT (add fresh)
7.5 μL
3.
Use 1-5mg (~ 10 cm – 50 cm) and cut hair into small pieces.
4.
Add sample to 0.2 PCR tube.
5.
Add 100 uL of 1x Lysis buffer and incubate for 5 minutes at room temperature.
6.
Centrifuge 14000 rpm for 5 minutes and remove the supernatant.
7.
Add 75 uL of Master Mix (based on Table 1) and mix well.
Note: Ensure the hair sample is completely covered by lysis buffer by a brief centrifuge.
8.
Incubate at 95°C for 2 hours or until the hair is wholly dissolved.
9.
Add 247.5 uL of 1x Binding buffer and mix well.
10.
Add 10 uL of Proteinase K (20 mg/ml), mix well, and incubate at 65°C for 30 minutes.
11.
Transfer all the solution to a 2 ml centrifuge tube.
12.
Centrifuge at 14000 rpm for 5 minutes and transfer the supernatant to a new 1.5 ml centrifuge tube.
13.
Immediately add 1.42 mL of 80% Isopropyl alcohol, 10 uL of BcMag™ HO-DNA Beads and mix well.
Note:
- Before pipetting, shake or Vortex the bottle to completely resuspend the Magnetic Beads.
- Do not allow the magnetic beads to sit for more than 2 minutes before dispensing.
14.
Incubate at room temperature for 15 minutes with gentle rotation.
15.
Place the tube on the magnetic Rack for 1-3 minutes. Remove the supernatant while the tube remains on the Rack. Add 500 uL of 85% Ethanol and wash the beads by slowly pipetting up and down 20-25 times. Again, place the tube on the magnetic Rack for 1-3 minutes and remove the supernatant completely while the tube remains on the Rack.
16.
Repeat step 13 once.
17.
Remove the tube from the magnetic Rack.
18.
Air dry for 30 minutes or dry them at 65°C for about 10 minutes (Ensure the Ethanol is completely evaporated).
19.
Add 10 uL of elution buffer for 1mg hair and 15 uL of elution buffer for 5mg hairs to elute DNA by slowly pipetting up and down 20-25 times.
20.
Place the tube on the magnetic Rack for 1-3 minutes. Transfer the supernatant to a new centrifuge tube while the tube remains on the Rack.
21.
Add 1µl of 10x Triton x-100 and 1µl 10x of NaCl to the eluted DNA mix well (Final concentration: 0.1% Triton x-100 and 0.5 M NaCl).
22.
Transfer 6µl BcMag™ Hair Pigment Removal Beads to a centrifuge tube and place the tube on the magnetic Rack for 1-3 minutes. Remove the supernatant while the tube remains on the Rack.
23.
Combine the beads with the DNA solution and mix by slowly pipetting up and down 20-25 times. Again, place the tube on the magnetic Rack for 1-3 minutes.
24.
Transfer the supernatant to a new centrifuge tube while the tube remains on the Rack.
25.
Store the extracted nucleic acids at -20°C.