Catalogue Number: PP-210L-JEN
|Shelf Life:||12 months|
|Type:||RNA Purification Kit|
Description: Total RNA Purification Kit is designed for rapid, high purity and high yield isolation of total RNA from small amounts of various samples including blood, animal and plant tissue, bacteria and viruses. The spin column based method allows complete removal of inhibitors such as divalent cations and proteins. Due to elimination of phenol, handling of the kit is safe and no harmful waste is produced. The purified total RNA can be used in a number of downstream applications. The kit allows the purification of up to 100 µg RNA per preparation.
Preparation procedure: Before start, add the following components (not included in the kit) as indicated on the respective bottle:2-Mercaptoethanol to the Lysis Buffer (10 µl 2-Mercaptoethanol per 1 ml Lysis Buffer)96-99 % Ethanol to Blood Washing Buffer, First Washing Buffer and Second Washing Buffer Buffer||PP-210XS10 preps||PP-210S50 preps||PP-210L250 preps Lysis Buffer||5.2 ml (add 52 µl 2-ME)||26 ml (add 260 µl 2-ME)||130 ml (add 1.3 ml 2-ME) Activation Buffer||1.2 ml||6 ml||30 ml BloodWashing Buffer||add 6.4 ml Ethanol (final volume 8 ml)||add 32 ml Ethanol (final volume 40 ml)||add 160 ml Ethanol (final volume 200 ml) First Washing Buffer||add 1.6 ml Ethanol (final volume 8 ml)||add 8 ml Ethanol (final volume 40 ml)||add 40 ml Ethanol (final volume 200 ml) Second Washing Buffer||add 6.4 ml Ethanol (final volume 8 ml)||add 32 ml Ethanol (final volume 40 ml)||add 160 ml Ethanol (final volume 200 ml) Elution Buffer||1 ml||5 ml||25 ml 1 Sample Preparation and Cell Lysis: BloodTransfer 100 µl of non-coagulating blood to a microcentrifuge tube.Add 500 µl of Lysis Buffer (2-ME added) and vortex for 10 sec. Fresh Tissue Sample - Animals or PlantsCollect 20-50 mg fresh tissue sample in a micro-centrifuge tube.Add 300 µl of Lysis Buffer (2-ME added) and homogenize the material using an appropriate apparatus (hand-operated pellet pestle or motor-driven grinder).Add additional 200 µl of Lysis Buffer (2-ME added) to the homogenized sample and vortex 15-30 sec (Note: Sample volume should not exceed 10 % of the Lysis Buffer volume).Centrifuge at 10,000 g for 10 min. Optional step in case that debris still remains in the supernatant:Add 500 µl chloroform (not included in the kit) and vortex for 15-30 sec.Centrifuge at 10,000 g for 10 min. Transfer the supernatant (if you added chloroform: the upper aqueous phase) into a microcentrifuge tube. Cells from Nasal or Throat SwabsAdd 500 µl of Lysis Buffer (2-ME added) to a microcentrifuge tube.Brush a sterile, single-use cotton swab or Buccal Swab Brush inside the nose or mouth of the subject.Cut the cotton tip where the nasal or throat cells were collected and place it into the microcentrifuge tube containing the Lysis Buffer (2-ME added).Close the tube, vortex and incubate at room temperature for 5 min. Cells Grown in MonolayerPut off culture media.Add 500 µl of Lysis Buffer (2-ME added) per 1-5 x 106 cells.Lyse cells and homogenize the sample by pipetting up and down several times. Cells Grown in SuspensionPellet 1-5 x 106 animal, plant or yeast cells, or 1 x 107 bacterial cells. (Occasionally, enzymatic lysis or mechanical disruption is required for cell-wall disruption of some yeast and bacterial cells.)Discard the supernatant and add 500 µl of Lysis Buffer (2-ME added).Lyse the sample by repetitive pipetting or vortexing for 10 sec. 2 Column Activation [optional]:Place a spin column into a 2 ml collection tube.Add 100 µl Activation Buffer into the Spin Column.Centrifuge at 10,000 g for 30 sec.Discard the