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MagBeads Plasma/Serum RNA+DNA Preparation Kit

Magnetic bead based purification of free circulating RNA and DNA from blood, serum or plasma

Cat. No. Amount Price (EUR) Buy / Note
PP-224S 50 preparations 133,50 Add to Basket/Quote Add to Notepad
PP-224L 5 x 50 preparations 534,00 Add to Basket/Quote Add to Notepad
Fig. 1: Schematic isolation procedure
Fig. 1: Schematic isolation procedure

For in vitro use only!

Shipping: shipped at ambient temperature

Storage Conditions: store at ambient temperature

Shelf Life: 12 months

Description:
MagBeads Plasma/Serum RNA+DNA Preparation Kit is developed for easy, efficient and fast isolation of free circulating RNA and DNA molecules (cNA) originating mostly from dead or destructed cells. Some cNA molecules are also synthesized by living cells and excreted into blood as part of the intercellular signaling system.
The analysis of such nucleic acids in blood allows the development of minimal invasive diagnostic approaches for screening of various clinical diseases and disorders. The presence of specific circulating RNA or DNA allows an early and reliable detection of pathological processes. This technique allows the diagnosis of early stages of cancer or viral infections, noninvasive prenatal screening during pregnancy or diagnosis of different forms of diabetes.

Sources of free DNA in blood are:

  • dead blood cells
  • dead pathogenic microorganisms (i.g. bacteria, viruses)
  • DNA molecules on the surface of leucocytes
  • apoptosis processes
  • necrosis processes
  • spontaneous release of newly synthesized DNA
  • spontaneous release of RNA/DNA-lipoprotein complexes from healthy cells
The level of free DNA in blood produced from dead cells and microorganisms is usually low. The main source of free DNA in blood is apoptosis. This is a typical process for oncological diseases.

Sources of free RNA are:

  • active or dead viruses circulating in blood
  • spontaneous release of RNA in form of RNA/DNA-lipoprotein complexes
  • mRNA from cells died from necrosis or apoptosis
The kit contains all necessary reagents in one box. The isolation procedure can be easily adapted for isolation of smaller or larger amounts of DNA.
The principle of the method is based on a reversible adsorption of DNA on magnetic particles coated with a special SiO2 surface. A schematic isolation procedure is shown on Figure 1. The sample cells are lysed and the containing DNA is bound to magnetic particles. After a repeated washing cycle the purified DNA is eluted from the magnetic particles.

Caution of Strong Magnetic Field
Magnetic fields can cause damage to magnetic storage media including credit cards, magnetic data tapes and computer hard drives. Strong magnetic fields can cause implanted heart pacemakers and cardioverter defibrillators to cease operation. Keep any electronic devices at least 1 Meter away from the magnets or magnetic racks. Interaction with metallic objects may produce pinch hazards.

Content:

ComponentPP-224S
50 Prep Kit
PP-224L
5 x 50 Prep Kit
Lysis Buffer6.5 ml5 x 6.5 ml
Binding Buffer20 ml5 x 20 ml
Magnetic particles RNA Grade250 μ5 x 250 μl
First Washing Buffer30 ml5 x 30 ml
Second Washing Buffer30 ml5 x 30 ml
Third Washing Buffer30 ml5 x 30 ml
Final Washing Buffer30 ml5 x 30 ml
Elution Buffer2.5 ml5 x 2.5 ml

Preparation procedure:
1 Before Starting

  • Resuspension of the magnetic particles is critical to achieve optimal results. Resuspend magnetic particles carefully by mixing or vortexing before use.
  • Check all buffers for precipitates. If a visible precipitation appears, place the buffer in a warm water bath (35-50°C) and shake it until it becomes clear. The formation of a precipitate does not affect the quality of buffers.
  • Mix all buffers well before use.


2 Preparation Volume

  • For isolation of larger sample volumes please increase the amounts of Lysis and Binding Buffer proportionally. All other buffer volumes have not to be changed.


Component100 μl sample volume500 μl sample volume
Lysis Buffer130 μl650 μl
Binding Buffer400 μl2 ml
Magnetic particles RNA Grade5 μl5 μl
First Washing Buffer600 μl600 μl
Second Washing Buffer600 μl600 μl
Third Washing Buffer600 μl600 μl
Final Washing Buffer600 μl600 μl
Elution Buffer50 μl50 μl

3 Sample Preparation and Cell Lysis

  • Place 100 μl of plasma or serum into a new 1.5 ml tube.
  • Add 130 μl well mixed Lysis Buffer and mix again by pipetting.
  • Incubate at room temperature for 5 min (longer incubation up to 15 min may increase DNA yield).


4 DNA Binding

  • Place 400 μl Binding Buffer and 5 μl of well mixed Magnetic Particles into a clean tube and mix both components thoroughly.
  • Add the prepared suspension of magnetic particles to the tube containing the prepared sample and mix thoroughly.
  • Incubate for 5 minutes at room temperature, mix once or twice during this time.
  • Place the tube for up to 1 minute in a magnetic rack to collect the magnetic particles.
  • Discard the supernatant without disturbing the magnetic particles at the tube wall.


5 First Washing

  • Place the tube in a non-magnetic rack.
  • Add 600 μl First Washing Buffer and mix thoroughly until a homogeneous suspension is obtained.
  • Place the tube in a magnetic rack to collect the magnetic particles.
  • Discard the supernatant without disturbing the magnetic particles at the tube wall.


6 Second Washing

  • Place the tube in a non-magnetic rack.
  • Add 600 μl Second Washing Buffer and mix thoroughly until a homogeneous suspension is obtained.
  • Place the tube in a magnetic rack to collect the magnetic particles.
  • Discard the supernatant without disturbing the magnetic particles at the tube wall.


7 Third Washing

  • Place the tube in a non-magnetic rack.
  • Add 600 μl Third Washing Buffer and mix thoroughly until a homogeneous suspension is obtained.
  • Place the tube in a magnetic rack to collect the magnetic particles.
  • Discard the supernatant without disturbing the magnetic particles at the tube wall.


8 Final Washing

  • Place the tube in a non-magnetic rack.
  • Add 600 μl Final Washing Buffer and mix thoroughly until a homogeneous suspension is obtained.
  • Place the tube in a magnetic rack to collect the magnetic particles.
  • Discard the supernatant without disturbing the magnetic particles at the tube wall.


9 DNA Elution

  • Incubate the tube in a thermal block at 60°C for 10 minutes to dry the pellet of magnetic particles.
  • Note: It is important to dry the beads since residual ethanol from washing buffer may interfere with downstream reactions.
  • Add 50 μl Elution Buffer and thoroughly resuspend the pellet by pipetting until a homogeneous suspension is obtained.
  • Note: If you wish to obtain higher DNA concentration use lower amounts of Elution Buffer.
  • Incubate the tube in a thermal block at 60°C for 10-15 minutes.
  • Place the tube in a magnetic rack to collect the magnetic particles.
  • Transfer the DNA-containing supernatant into a fresh tube without disturbing the magnetic particles at the tube wall.
  • The ultra-pure genomic DNA is ready for further use.
  • Note: Store the isolated DNA at -20°C.


Troubleshouting
Purity of isolated nucleic acid is determined by the ratio of optical densities at 260 and 280 nm wavelength. The OD260/OD280 ratio must be in the range of 1.7-2.0.

ProblemPossible
Reasons
Solution
Low nucleic acid yieldA. Sample condition
1. Not enough DNA-containing cells were collected.
2. The collected material was too old or faced to many freeze-thawing cycles.

1. Use more source material or elute DNA in a smaller volume of Elution Buffer.
2. Repeat material collection procedure.
B. Magnetic Particles were not dried thoroughly enough before the DNA elution.Increase the drying time of the magnetic particle pellet after the Final Wash step.
C. Lysis process not completed.Mix the sample strongly after adding Lysis Buffer.
D. Too big volume of Elution buffer added.Find optimal buffer volume to achieve the required concentration of DNA.
OD260/OD280 ratio is too lowHigh protein impurity1. Try to achieve better resuspensions of magnetic particles on each step of the isolation procedure.
2. Use smaller amount of particles or bigger volume of Elution buffer.
Isolated DNA seems degradedThe sample material was too old or faced to many freeze-thawing cycles.Repeat material collection procedure. Avoid freezing samples during transportation and storage.