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Protocol

One-Step Dandruff Cell DNA Purification Protocol

Products

BcMag™ One-Step Dandruff Cell DNA Purification Kit
Cat. No.  AAA101

Unit Size  50x preps
Order
BcMag™ One-Step Dandruff Cell DNA Purification Kit
Cat. No.  AAA102

Unit Size  100x preps
Order
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Components

BcMag™ U-DNA Beads
Storage  4°C

Cat. No. AAA-101 (50 Preps)
2.5 ml

Cat. No. AAA-102 (100 Preps)
5.0 ml
10x Lysis Buffer (100mM Tris-HCl, PH 9.0)
Storage  4°C

Cat. No. AAA-101 (50 Preps)
0.6 ml

Cat. No. AAA-102 (100 Preps)
1.2 ml
Proteinase K
Storage  -20°C

Cat. No. AAA-101 (50 Preps)
12.5 mg

Cat. No. AAA-102 (100 Preps)
25 mg
DTT
Storage  -20°C

Cat. No. AAA-101 (50 Preps)
15.4 mg

Cat. No. AAA-102 (100 Preps)
30.8 mg
Proteinase K Suspension Buffer
Storage  4°C

Cat. No. AAA-101 (50 Preps)
1.0 ml

Cat. No. AAA-102 (100 Preps)
2.0 ml
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Shipping conditions: At ambient temperature

Handling And Storage: Store The Kit Components According To The Table Above On Arrival.

Cat. No.

AAA101

AAA102

Product Name

BcMag™ One-Step Dandruff Cell DNA Purification Kit

BcMag™ One-Step Dandruff Cell DNA Purification Kit

Unit Size

50x preps

100x preps

Order

Components

BcMag™ U-DNA Beads

10x Lysis Buffer (100mM Tris-HCl, PH 9.0)

Proteinase K

DTT

Proteinase K Suspension Buffer

Storage

4°C

4°C

-20°C

-20°C

4°C

Cat. No. AAA101 (50 Preps)

2.5 ml

0.6 ml

12.5 mg

15.4 mg

1.0 ml

Cat. No. AAA102 (100 Preps)

5.0 ml

1.2 ml

25 mg

30.8 mg

2.0 ml

Shipping conditions: At ambient temperature

Handling and Storage: Store the kit components according to the table Above on arrival.

Introduction

BcMag™ One-Step Dandruff Cell DNA Purification protocol is designed to streamline and enhance the process of genomic DNA purification from Dandruff. The protocol avoids the laborious and time-consuming bind-wash-elute method with its risk of DNA loss and buffer carryover. With BcMag™ One-Step DNA purification protocol, a negative selection chromatography approach utilizing magnetic beads effectively captures and eliminates impurities such as PCR inhibitors, while preserving the DNA. This results in a fast, simple and efficient DNA extraction with no need for carrier RNA, no liquid transfer and minimal equipment required. Imagine processing hundreds of samples in less than 30 minutes with ease and confidence.

PCR inhibitor removal by magnetic beads

Principle

The specially designed magnetic beads with our proprietary surface chemistry function capture the impurity once mixed with the sample. The magnetic beads-impurity complex is then magnetically removed by a magnet while the pure DNA remains in the solution.

Workflow of One-Step Dandruff Cell DNA Purification Kit

1.

Add functional magnetic beads to the sample.

2.

Mix the samples with the magnetic beads and proteinase K to lyse the cells.

3.

Mix by vortexing/pipetting for the beads to capture the PCR inhibitors.

4.

Remove the beads with a magnet.

5.

Aspirate the supernatant containing the pure ready-to-use DNA/RNA.

Performance

The purified DNA is suitable for use in sensitive downstream applications, such as PCR, qPCR, single-nucleotide polymorphism (SNP), short tandem repeat (STR) genotyping, genotyping, or next-generation sequencing (NGS), buccal DNA sample collection, veterinary genotyping and diagnostics, research genotyping, Veterinary genotyping and diagnostics, genetic testing, forensics, population studies.

Features and Advantages

Rapid and efficient extraction protocol: without prior DNA isolation for subsequent use in direct workflows, No liquid transfer, and One-tube.

Ultrafast: Process 96 samples in less than an hour.

Highest nucleic acids recovery rates: Minimal loss of DNA during extraction

Effectively removes inhibitors: polyphenolic compounds, humic/fulvic acids, acidic polysaccharides, tannins, melanin, heparin, detergents, denim dyes, divalent cations such as Ca2+, Mg2+, etc.

Cost-effective: Eliminates columns, filters, laborious repeat pipetting, and organic reagents.

High throughput: Compatible with many different automated liquid handling systems.

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)

Since there is no concentration step in the protocol, the concentration of the nucleic acid depends on the quality and quantity of the sample used.

Quantification of the nucleic acids: Use only fluorescence methods such as qPCR, Qubit, and Pico Green.

OD260 methods such as Nanodrop and UV-spectrophotometry are not-suitable.

For long-term storage, store the extracted nucleic acids at -20°C.

Materials Required by the User

Item

Magnetic Rack for centrifuge tube


** Based on sample volume, the user can choose one of the following magnetic Racks

Source

•  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)

Item

BcMag™ 96-well Plate Magnetic Rack.

Source

•  BcMa™ 96-well Plate Magnetic Rack (side-pull) compatible with 96-well PCR plate and 96-well microplate or other compatible Racks (Bioclone, Cat. No. MS-06)

Item

Adjustable Single and Multichannel pipettes

Item

Centrifuge with swinging bucket

Addition items are required if using 96-well PCR plates / tubes

Vortex Mixer

** The user can also use other compatible vortex mixers. However, the Time and speed should be optimized, and the mixer should be: Orbit ≥1.5 mm-4 mm, Speed ≥ 2000 rpm

Eppendorf™ MixMate™

Eppendorf, Cat. No. 5353000529

Tube Holder PCR 96

Eppendorf, Cat. No. 022674005

Tube Holder 1.5/2.0 mL, for 24 × 1.5 mL or 2.0 mL

Eppendorf, Cat. No. 022674048

Smart Mixer, Multi Shaker

BenchTop Lab Systems, Cat. No. 5353000529

1.5/2.0 mL centrifuge tube

96-well PCR Plates or 8-Strip PCR Tubes

PCR plates/tube

! IMPORTANT ! If using other tubes or PCR plates, make sure that the well diameter at the bottom of the conical section of PCR Tubes or PCR plates must be ≥2.5mm.

Items

Magnetic Rack for centrifuge tube

** Based on sample volume, the user can choose one of the following magnetic Racks

Source

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)

BcMag™ 96-well Plate Magnetic Rack

BcMa™ 96-well Plate Magnetic Rack (side-pull) compatible with 96-well PCR plate and 96-well microplate or other compatible Racks (Bioclone, Cat. No. MS-06)

Adjustable Single and Multichannel pipettes

Centrifuge with swinging bucket

Addition items are required if using 96-well PCR plates/tubes

Vortex Mixer

** The user can also use other compatible vortex mixers. However, the Time and Speed should be optimized, and the mixer should be: Orbit ≥1.5 mm-4 mm, Speed ≥ 2000 rpm

Eppendorf™ MixMate™

Tube Holder PCR 96

Tube Holder 1.5/2.0 mL, for 24 × 1.5 mL or 2.0 mL

Smart Mixer, Multi Shaker

Eppendorf, Cat. No. 5353000529

Eppendorf, Cat. No. 022674005

Eppendorf, Cat. No. 022674048

BenchTop Lab Systems, Cat. No. 5353000529

Eppendorf™ MixMate™

Tube Holder PCR 96

Tube Holder 1.5/2.0 mL, for 24 × 1.5 mL or 2.0 mL

Smart Mixer, Multi Shaker
 

Eppendorf, Cat. No. 5353000529

Eppendorf, Cat. No. 022674005

Eppendorf, Cat. No. 022674048
 

BenchTop Lab Systems, Cat. No. 5353000529

1.5/2.0 mL centrifuge tube

96-well PCR Plates or 8-Strip PCR Tubes

PCR Plates/Tubes

! IMPORTANT ! If using other tubes or PCR plates, make sure that the well diameter at the bottom of the conical section of PCR Tubes or PCR plates must be ≥2.5mm.

A. Sample Preparation

a.

Use sterile ophthalmic forceps to pick up single pieces of dandruff (up to 5mg) and place them in a clean PCR tube.

b.

When using forensic lifting tape, isolate single pieces of dandruff with a diameter greater than 500 µm from the tapes with a stereomicroscope and sterile ophthalmic forceps and place them in a clean PCR tube.

B. Premix Beads Solution Preparation

! IMPORTANT !

1.

Before pipetting, shake or Vortex the bottle to completely resuspend the Magnetic Beads.

2.

Do not allow the magnetic beads to sit for more than 2 minutes before dispensing.

3.

Proteinase K preparation: Provide protease K as lyophilized powder and dissolve at a 20 mg/ml concentration in Proteinase K Suspension Buffer. For example, 12.5 mg dissolved in 625 µl 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.

4.

4. DTT solution preparation: Provide DTT as powder and dissolve at a concentration of 1M in dH2O. For example, 15.4 mg dissolved in 100µl 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.

5.

Dilute DTT to a concentration of 10 mM from stock with dH2O and use it immediately. Discard unused DTT solution.

6.

Prepare a fresh Master Mix following Table 2 for the number of samples to be processed, plus 10% more (e.g, if you have 10 samples, prepare a Master Mix for 11). Add the following components to the reservoir.

Table 2. Premix Beads solution

Components

BcMag™ U-DNA Beads

10x Lysis Buffer

Proteinase K (20mg/ml)

DTT (10 mM)

Sample

Ultrapure Water

Total

One Well ( 100 μL Reaction Volume)

50 μL

10 μL

12.5 μL

3 μL

x

x

100 μL

Components

BcMag™ U-DNA Beads
One Well ( 100 μL Reaction Volume)

50 μL
10x Lysis Buffer
One Well ( 100 μL Reaction Volume)

10 μL
Proteinase K (20mg/ml)
One Well ( 100 μL Reaction Volume)

12.5 μL
DTT (10 mM)
One Well ( 100 μL Reaction Volume)

3 μL
Sample
One Well ( 100 μL Reaction Volume)

x
Ultrapure Water
One Well ( 100 μL Reaction Volume)

x
Total
One Well ( 100 μL Reaction Volume)

100 μL
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C. Isolation Procedure

! IMPORTANT !

Pipet up and down premix beads solution in a reagent reservoir until the solution is homogeneous before dispensing.

Do not allow the magnetic beads to sit for more than 5 minutes before dispensing.

1.

Transfer the appropriate amount of premix beads solution (Table 2) to the sample. (For example, 100µl for 5mg of dandruff, 30 µl for 1 mg of dandruff).

2.

Mix the sample well by pipetting

3.

Place the PCR plate/tube into a thermocycler and incubate at:

a.

65°C for 15 minutes

b.

80°C for 10 minutes

4.

Remove the PCR plate/tube from the thermocycler and mix the sample with beads by slowly pipetting up and down 20-25 times or Vortex the sample at 2000 rpm for 5 minutes (see picture).

VortexSample

5.

Centrifuge at 3500 rpm for 5 minutes.

6.

Place the sample plate/tube on the magnetic separation plate for 30 seconds or until the solution is clear.

7.

Transfer the supernatant to a clean plate /tube while the sample plate remains on the magnetic separation plate. The sample is ready for downstream applications. Using 1-5 μl in a 25μl RT-PCR or qPCR.

D. Troubleshooting

Problem

Low DNA/RNA Recovery

Probable Cause

Poor starting sample material

Suggestion

  • Use better-quality samples.
  • Add more samples

Problem

Ct Value Delays

Probable Cause

Too many PCR inhibitors in the sample.

Suggestion

  1. Add 25-50 μL BcMag™ U-DNA Beads to the extract solution and mix by slowly pipetting up and down 20-25 times or Vortex the sample at 2000 rpm for 5 minutes. Place the sample plate/tube on the magnetic separation plate for 30 seconds or until the solution is clear.
  1. Transfer the supernatant to a clean plate/tube while the sample plate remains on the magnetic separation plate. Using 1-5 μl in a 25μl RT-PCR or qPCR. The sample is ready for downstream applications.

Problem

Ct Value Delays

Probable Cause

Recovery DNA is so low.

Suggestion

  • Use better-quality samples.
  • Add more samples

Problem

Probable Cause

Suggestion

Low DNA/RNA Recovery

Poor starting sample material

  • Use better-quality samples.
  • Add more samples

Ct Value Delays

Too many PCR inhibitors in the sample.

  1. Add 25-50 μL BcMag™ U-DNA Beads to the extract solution and mix by slowly pipetting up and down 20-25 times or Vortex the sample at 2000 rpm for 5 minutes. Place the sample plate/tube on the magnetic separation plate for 30 seconds or until the solution is clear.
  1. Transfer the supernatant to a clean plate/tube while the sample plate remains on the magnetic separation plate. Using 1-5 μl in a 25μl RT-PCR or qPCR. The sample is ready for downstream applications.

Recovery DNA is so low.

  • Use better-quality samples.
  • Add more samples

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