Protocol
BcMag™ Bone and Teeth DNA Purification Protocol
Products
Components
BcMag™ HO-DNA Beads
1x Lysis Buffer
1x Elution Buffer
Proteinase K (20mg/ml)
Proteinase K Suspension Buffer
1 M DTT
Storage
4°C
4°C
4°C
-20°C
4°C
-20°C
Cat. No. AB101 (50 Preps)
0.75 ml
5.0 ml
1.5 ml
10 mg
0.5 ml
77 mg
Cat. No. AB102 (100 Preps)
1.5 ml
10 ml
3.0 ml
20 mg
1.0 ml
154 mg
Handling and Storage: Store the kit components according to the table below on arrival.
With the BcMag™ Bone and Teeth DNA Purification Kit, users can expect consistent, high-quality results every time. The kit is also user-friendly, with clear instructions and an easy-to-follow protocol, making it ideal for both novice and experienced users. Additionally, the kit comes with all the necessary reagents, including magnetic beads, buffers, and proteinase K, eliminating the need to purchase additional components.
Another important aspect of the BcMag™ Bone and Teeth DNA Purification Kit is its specificity. The kit is specifically designed to isolate DNA from bone and teeth specimens, ensuring optimal results and reducing the chance of cross-contamination with other types of samples. This specificity also reduces the risk of co-purifying inhibitors, which can interfere with downstream applications.
The purified DNA obtained using the BcMag™ Bone and Teeth DNA Purification Kit is highly suitable for a variety of applications, including STR analysis, gene sequencing, and more. The exceptional quality of the DNA means that users can expect high-quality results in these applications, without the need for extensive cleanup procedures.
In conclusion, the BcMag™ Bone and Teeth DNA Purification Kit is an essential tool for any laboratory that needs to isolate DNA from bone and teeth specimens. Its high yield and exceptional quality, combined with its ease of use and specificity, make it the ideal choice for researchers in both academia and industry.The BcMag™ Bone and Teeth DNA Purification Kit has been expertly designed to extract total nucleic acids from bone and teeth specimens with ease and efficiency. Utilizing our proprietary magnetic beads technology, in conjunction with a specially formulated demineralization buffer, this kit provides superior yield and exceptional quality of DNA. The purified genomic DNA obtained is of the highest integrity and can be utilized in a multitude of downstream applications, such as qPCR and STR analysis.
The isolation protocol of the Bone-Teeth DNA Purification Kit employs gentle lysis conditions, eliminating harsh methods such as alkaline lysis and the use of toxic chemicals. This helps to maintain the integrity of the DNA and eliminates the need for time-consuming cleanup procedures using organic solvents.
The workflow for Bone-Teeth DNA Isolation is as follows:
1.
Add the lysis buffer and proteinase K to the sample and incubate at 65°C to lyse the bone.
2.
Mix the functional magnetic beads with the sample and vortex or pipette the mixture.
3.
Wash the magnetic beads.
4.
Use a magnet to separate the beads from the sample.
5.
Elute the purified DNA from the magnetic beads.
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)
●
DNA size: Varies (depends on the quality of starting material
●
For long-term storage, store the extracted nucleic acids at -20°C.
●
Proteinase K preparation: Provide protease K as lyophilized powder and dissolve at a 20 mg/ml concentration in 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 dissolve at a concentration of 1M in dH2O. For example, 77 mg dissolved in 500µl dH2O. 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.
A. Materials Required by the User
●
95–100% ethanol
●
80% isopropyl alcohol
●
65°C Incubator chamber
●
Microcentrifuge tubes, 1.5ml
●
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)
B. Sample collection
The quality of an STR profile derived from a bone sample is determined by the bone’s type, age, and environmental storage state. The quality of DNA is greatly influenced by soil and humidity conditions. The success of purifying nuclear DNA from bone is also dependent on DNA integrity.
Finding good sampling locations in bones is exceptionally problematic. When working with cremated remains, materials must be handled to reduce contaminants while maintaining adequate material for DNA extraction. Incorrect extraction handling and sample storage might result in cross-contamination of target DNA with non-target DNA. It has been proposed that bone density influences DNA yield because denser tissues give higher physical protection from harm. As a result, DNA is frequently extracted from teeth protected by strong enamel or dense, weight-bearing long bones (tibia or femur). The petrous (or petrosal) part of the temporal bone is denser than many other skeletal sites and has been demonstrated to give much more usable DNA, sometimes four to sixteen times more than teeth.
To effectively extract DNA from the calcium matrix, bone must be preprocessed by removing and discarding the bone or teeth surfaces using scalpels and forceps. The extraction process’s success depends on the degree of grinding, which can be performed through physical grinding or using a low-speed drill to reduce heat buildup. The extraction procedure works best with finely ground bone because cells dispersed in the bone matrix are more accessible for lysis.
C. Purification
1.
Add 10mg of pulverized bone powder into 1.5ml tubes.
2.
Make bone lysis cocktail according to the instructions below, allowing for an excess of n+2 samples:
Components
1x Lysis Buffer
Proteinase K (20mg/ml)
1 M DTT
Total
100 μL reaction volume for 5 mg bone
3.
Add 100 μL of lysis cocktail to each 1.5ml tube containing the bone powder.
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)
●
DNA size: Varies (depends on the quality of starting material)
●
For long-term storage, store the extracted nucleic acids at -20°C.
●
Proteinase K preparation: Provide protease K as lyophilized powder and dissolve at a 20 mg/ml concentration in 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 dissolve at a concentration of 1M in d2H2O. For example, 77 mg dissolved in 500µl d2H2O. 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.
●
95–100% ethanol
●
80% isopropyl alcohol
●
65°C Incubator chamber
●
Microcentrifuge tubes, 1.5ml
●
Aerosol-resistant micropipette tip
●
Magnetic rack: Based on sample volume, the user can choose one of the following magnetic Separators:
BcMag™ separator-2 for holding two individual 1.5 ml centrifuge tubes (Bioclone, Cat.# MS-01)
BcMag™ separator-6 for holding six individual 1.5 ml centrifuge tubes (Bioclone, Cat.# MS-02)
BcMag™ separator-24 for holding twenty-four individual 1.5-2.0 ml centrifuge tubes (Bioclone, Cat.# MS-03)
BcMag™separator-50 for holding one 50 ml centrifuge tube, one 15 ml centrifuge tube, and four individual 1.5 ml centrifuge tubes (Bioclone, Cat.# MS-04)
B. Sample Collection
The quality of an STR profile derived from a bone sample is determined by the bone’s type, age, and environmental storage state. The quality of DNA is greatly influenced by soil and humidity conditions. The success of purifying nuclear DNA from bone is also dependent on DNA integrity.
Finding good sampling locations in bones is exceptionally problematic. When working with cremated remains, materials must be handled to reduce contaminants while maintaining adequate material for DNA extraction. Incorrect extraction handling and sample storage might result in cross-contamination of target DNA with non-target DNA. It has been proposed that bone density influences DNA yield because denser tissues give higher physical protection from harm. As a result, DNA is frequently extracted from teeth protected by strong enamel or dense, weight-bearing long bones (tibia or femur). The petrous (or petrosal) part of the temporal bone is denser than many other skeletal sites and has been demonstrated to give much more usable DNA, sometimes four to sixteen times more than teeth.
To effectively extract DNA from the calcium matrix, bone must be preprocessed by removing and discarding the bone or teeth surfaces using scalpels and forceps. The extraction process’s success depends on the degree of grinding, which can be performed through physical grinding or using a low-speed drill to reduce heat buildup. The extraction procedure works best with finely ground bone because cells dispersed in the bone matrix are more accessible for lysis.
C. Purification
1.
Add 10mg of pulverized bone powder into 1.5ml tubes.
2.
Make bone lysis cocktail according to the instructions below, allowing for an excess of n+2 samples:
Components
1x Lysis Buffer
Proteinase K (20mg/ml)
1 M DTT
Total
100 μL reaction volume for 5 mg bone
3.
Add 100 μL of lysis cocktail to each 1.5ml tube containing the bone powder.
4.
Mix the sample by pipetting.
5.
Incubate the tubes in the Incubator chamber with a gentle shake at 65°C for 24 hours.
6.
Remove the tubes from the chamber and mix by vortex or pipetting.
7.
Centrifuge the tubes at 12,000 × g for 5 minutes.
8.
Carefully transfer supernatant to a new 1.5ml centrifuge tube.
9.
Transfer 20 μL of supernatant to a new 1.5ml centrifuge tube.
10.
Add 97 μL of 80% isopropyl alcohol and mix by vortex or pipetting.
11.
Add 15 μLof BcMag™ HO-DNA Beads and mix by vortex or pipetting.
Note:
●
Vigorously shake the bottle until the magnetic beads become homogeneous before dispensing. Do not allow the beads to sit for more than 2 minutes before dispensing. Resuspend the magnetic beads every 2 minutes.
12.
Incubate at room temperature for 15 minutes with gentle rotation.
13.
Place the tube on the magnetic separator for 1-3 minutes. Remove the supernatant while the tube remains on the separator. Add 200μLof 85% Ethanol and mix by pipetting 10-15 times to wash the beads. Place the tube on the magnetic separator for 1-3 minutes and remove the supernatant completely while the tube remains on the separator.
14.
Repeat step (13) two times.
15.
Remove the tube from the magnetic separator and let the beads air dry for 10-30 minutes to evaporate the ethanol completely.
16.
Add 15μL to 30μLof 1x Elution buffer and mix by pipetting 30 times to elute the DNA from the beads. Place the tube on the magnetic separator for 1-3 minutes and transfer the supernatant to a new centrifuge tube.
17.
The eluted DNA should be stored at -20°C.
D. Troubleshooting
Problem
Low DNA Recovery
Probable Cause
Poor starting sample material
Suggestion
- Use better quality of the sample.
- Add more samples
■ Explore
■ Documents
Instruction Manual
• Bone-Teeth DNA Purification Kit Protocol (PDF)
■ Safety Data Sheets (SDS)
• SDS-DNA Purification