INTENDED USE OF HALO™ MagKit
HALO™ MagKit is designed for isolation of total DNA/RNA from clinical material: smears, scrapings and mucousa of the urogenital, respiratory and digestive tracts, biological fluids (plasma, serum, saliva, detachable conjunctiva, cerebrospinal fluid, pleural, synovial and amniotic fluids). Isolated DNA/RNA is suitable for further molecular biological studies, including RT-PCR.
PERFORMANCE OF HALO™ MagKit 100 REAGENTS
HALO™ MagKit 100 is a highly sensitive kit that allows efficient isolation both RNA and DNA from clinical material. The sensitivity limit is 5 genome equivalents per extraction.
HALO™ MagKit 100 allows to conduct both standard and high sensitivity studies with a sample volume of up to 100 μl.
HALO™ MagKit 100 allows to purify RNA/DNA both in manual mode (on a magnetic separation stand or using a centrifuge) and in automatic mode using open robotic stations.
Purification of RNA/DNA using HALO™ MagKit 100 is simple and convenient and does not require additional reagents.
HALO™ MagKit 100 set of reagents is designed to conduct studies of 100 samples with a volume of 100 μl (table 1).
Table 1. The component composition of HALO™ Mag kit
|Name of component||Volume|
|1. Magnetic sorbent||1 tube – 1.6 ml|
|2. Sorbing solution||1 bottle – 45 ml|
|3. Wash solution 1||1 bottle – 50 ml|
|4. Wash solution 2||1 bottle – 50 ml|
|5. Elution solution||1 bottle – 15 ml|
PRINCIPLE OF DNA/RNA EXTRACTION WITH HALO™ Mag KIT
DNA/RNA isolation method with HALO™ MagKit 100 is based on nucleic acid binding with magnetic sorbent in the presence of chaotropic salts and their subsequent elution into a low-salt buffer.
When working with HALO™ Mag kit’s reagents, it is necessary to observe the safety measures established for the appropriate sphere of clinical laboratory of PCR diagnostics.
REQUIRED EQUIPMENT AND MATERIALS
— laminar box or PCR-box;
— thermomixer, heated orbital incubator, heating block or water bath for microcentrifuge tubes with heating temperature up to 65 °С;
— microcentrifuge for tubes with rotation speed not less than 12 000 rpm/min;
— pipets with variable volume (10 – 1000 µl);
— pipet tips (to avoid cross-contamination, we recommend pipet tips with aerosol barriers);
— 1.5 ml or 2 ml microcentrifuge tubes;
— 1.5 ml or 2 ml microcentrifuge tubes rack;
— refrigerator with freezing camera (temperature from -24 °С to +8 °С);
— disposable gloves;
— waist tank;
— workstation disinfectant kit.
When working with RNA/DNA, it is necessary to use consumables that have a special marking “RNAse-free”, “DNAse-free”.
PREPARTION OF THE TEST SAMPLES
Collection, transportation and storage of samples is carried out in accordance with applicable rules and instructions.
Samples of urine are requiring pre-processing in accordance with generally accepted methodological recommendations.
All other samples are to be thoroughly homogenized with a vortex immediately prior to DNA/RNA extraction; drops of material from the inside of the lid are removed by short-term centrifugation at minimum speeds.
RNA/DNA EXTRACTION FROM 100 µL SAMPLE USING MAGNETIC SEPARATION STAND
0. If Sorbing solution and/or Wash solution 1 contains precipitate, it needs to be dissolved by heating to 55-60°C with gentle agitation. Carefully re-suspend the Magnetic sorbent on the vortex for the period of 10 s before starting extraction procedure.
I. Prepare the mixtures of Sorbing solution, Magnetic sorbent and Internal extraction control (in case of use) at the rate of 450 μl of Sorbing solution, 16 μl of Magnetic sorbent and 10 μl of Internal extraction control (when using) for one isolation at container of the corresponding volume. Mix the resulting mixture on a vortex. Table 2 shows the calculation of the component’s volumes according to certain samples quantity. It is recommended to add the entire volume of the Magnetic sorbent and Internal extraction control into a bottle with a Sorbing solution if making one-time extraction of 100 samples.
Table 2. The Ratios of components.
|The quantity of samples||Sorbing solution (ml)||Magnetic sorbent (ml)||Internal extraction control (ml)||The quantity of samples||Sorbing solution (ml)||Magnetic sorbent (ml)||Internal extraction control (ml)|
II. Pipette 466 μl or 476 μl (if using internal control) of the prepared mixture (step I) into 1.5 or 2 ml microcentrifuge tube.
III. Add 100 µl of the sample in each tube (see page 1, PREPARTION OF THE TEST SAMPLES).
IV. Mix tubes content by pulse-vortexing for 5-10 s and incubate at 65°C for 5 min with constant (recommended) or periodical mixing (pulse-vortexing for 2 times).
V. Remove drops from the inside of the lid by short-term centrifugation at the minimum speed.
VI. Place tubes on the magnetic separation stand for 30-60 s period. Carefully remove supernatant avoiding touching the magnetic sorbent.
VII. Add 500 µl of Wash solution 1 and mix tubes content by pulse-vortexing for 5-10 s.
VIII. Remove drops from the inside of the lid by short-term centrifugation at minimum speeds.
IX. Transfer tubes on a magnetic separation stand for 30-60 s. Carefully remove supernatant avoiding touching the magnetic sorbent.
X. Add 500 µl of Wash solution 2 and mix tubes content by pulse-vortexing for 5-10 s.
XI. Remove drops from the inside of the lid by short-term centrifugation at minimum speed.
XII. Place tubes on the magnetic separation stand for 30-60 s period. Carefully remove supernatant without touching the magnetic sorbent.
At this stage, it is necessary to remove cautiously the residue of Wash Solution 2, because it can cause inhibition of PCR.
XIII. Add 100 µl of Elution solution and mix tubes content by pulse-vortexing for 5-30 s.
XIV. Incubate the tubes at 65°C for 5 min with constant (recommend) or periodical mixing (pulse-vortexing for 5-10 s every 2 minutes). After the incubation end mix tubes by pulse-vortexing for 2 s.
XV. Remove drops from the inside of the lid by short-term centrifugation at minimum speed.
XVI. Place tubes on the magnetic separation stand for 30-60 s. Supernatant contains purified RNA and DNA ready for reverse transcription and PCR.
It is strongly recommended to transfer the supernatant to the new tubes for long-term sample storage. Storage of samples is allowed for the period of 24 hours at the temperature of 4 °C or for 1 year at a temperature of -16 °C.
TRANSPORTATION AND STORAGE OF HALO™ Mag KIT
Transportation period of HALO™ MagKit 100 is 3 days at normal ambient temperature (but not more than 30 °C).
HALO™ MagKit 100 may be transported by any type of transport in conditions ensuring it safety, in accordance with the rules for the goods carriage for this type of transport.
HALO™ MagKit 100 is stored at the manufacturer’s packaging at +2 — +8 °C temperature for the entire shelf life.
The shelf life of HALO™ MagKit 100 is 12 months from the date of manufacture.