RNA/DNA/Protein Purification Plus Kits
For research use only and NOT intended for in vitro diagnostics.
Formerly Cat. 24200, 24210, and 24000
RNA/DNA/Protein Purification Plus Kits
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Supporting Data
Figure 1. Recovery of True Total RNA including microRNA from HEK-293 Cells. Panel A is a 1X MOPS 1% agarose gel showing the RNA that was isolated from 2 different samples of ~ 800,000 HEK-293 cells using either Norgen's RNA/DNA/Protein Purification Plus Kit or a competitor's multiple-analyte purification kit. Both kits isolated large RNA (represented by 28S and 18S rRNA) with high integrity but Norgen's RNA/DNA/Protein Purification Plus Kit provided the added benefit of recovering small RNA without any additional protocol. Panel B is a result from a bioanalyzer resolution of the eluted RNA. Similar to the agarose gel, Norgen's RNA/DNA/Protein Purification Plus Kit showed the added benefit of recovering small RNA. The difference in small RNA recovery was also demonstrated by gene-specific RT-qPCR. One microgram of RNA was used in RT-qPCR reactions for human S15 (for Large RNA) and miR-21 (for microRNA) genes. The RNA isolated by Norgen's RNA/DNA/Protein Purification Plus Kit showed similar Ct value to RNA isolated by the competitor's kit for the large RNA (Panel D). However, Norgen's RNA/DNA/Protein Purification Plus Kit showed superior recovery of small RNA including microRNAs as depicted by the miR-21 RT-qPCR (Panel C).
Figure 2. Recovery of True Total RNA including microRNA from Hamster Liver. Panel A is a 1X MOPS 1% agarose gel showing the RNA that was isolated from 2 different samples of 15 mg hamster liver using either Norgen's RNA/DNA/Protein Purification Plus Kit or a competitor's multiple-analyte purification kit. Both kits isolated large RNA (represented by 28S and 18S rRNA) with high integrity but Norgen's RNA/DNA/Protein Purification Plus Kit provided the added benefit of recovering small RNA without any additional protocol. Panel B is a result from a bioanalyzer resolution of the eluted RNA. Similar to the agarose gel, Norgen's RNA/DNA/Protein Purification Plus Kit showed the added benefit of recovering small RNA. The difference in small RNA recovery was also demonstrated by gene-specific RT-qPCR. One microgram of RNA was used in RT-qPCR reactions for hamster beta-Actin (for Large RNA) and miR-21 (for microRNA) genes. The RNA isolated by Norgen's RNA/DNA/Protein Purification Plus Kit showed similar Ct value to RNA isolated by the competitor's kit for the large RNA (Panel D). However, Norgen's RNA/DNA/Protein Purification Plus Kit showed superior recovery of small RNA including microRNAs as depicted by the miR-21 RT-qPCR (Panel C).
Figure 3. Recovery of Intact, High Quality Genomic DNA from HEK-293 cells and Hamster Liver. Panel A is a 1% agarose gel showing the gDNA isolated from the same HEK-293 cell or hamster liver samples using Norgen's RNA/DNA/Protein Purification Plus Kit or competitor's multiple-analyte purification kit. Lane M is Norgen's HighRanger 1 kb DNA Ladder and the sample lanes contain 10 µL of each of the 100 µL elutions. The gel showed high quality, and intact genomic DNA, with a better yield using Norgen's RNA/DNA/Protein Purification Plus Kit. Panel B is the result of qPCR amplification of 25 ng of eluted genomic DNA using 5S rRNA-specific primers. Genomic DNA isolated using Norgen's RNA/DNA/Protein Purification Plus Kit is of high quality and performed similar to or better than competitor's product.
Figure 4. High Quality Total Proteins Eluted in Mass Spec-Compatible Buffer. Norgen's RNA/DNA/Protein Purification Plus Kit provides an additional column purification step for effective concentration and clean-up of the isolated proteins. In contrast, most competing multiple analyte isolation products require protein precipitation and the precipitated proteins are required to be resuspended in buffer with high-detergent content (such as SDS-PAGE loading dye) for full recovery. Protein fractions (from hamster liver) isolated by Norgen's RNA/DNA/Protein Purification Plus Kit and a competitor's kit were resolved on a 12% SDS-PAGE protein gel. Panel A showed that when the competitor's precipitated protein fraction was resuspended in a provided SDS-PAGE loading buffer, the protein recovery was similar among the two kits. Panel B showed that when the same precipitated protein fraction from the competitor's kit was resuspended in a Tris-based buffer containing no detergent or denaturant, the protein recovery became drastically reduced. In contrary, Norgen's RNA/DNA/Protein Purification Plus Kit purified proteins by column and the eluted proteins are already in a buffer compatible with most downstream applications including mass spectrophotometry as well as standard protein quantification methods (including Bradford assays).
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Kit Specifications
|
|
| Maximum Column Binding Capacity |
50 μg for RNA
20 μg for DNA 200 μg for protein |
| Maximum Column Loading Volume |
650 μL
|
| Size of RNA Purified |
All sizes, including small RNA
(< 200 nt) |
| Size of DNA Purified |
≥ 30 kb
|
| Maximum Amount of Starting Material: Animal Cells Animal Tissues Blood Bacteria Yeast Fungi Plant Tissues |
5 x 106 cells 25 mg (for selected tissues) 100 μL 1 x 109 cells 1 x 108 cells 50 mg 50 mg |
| Time to Complete 10 Purifications |
30 minutes
|
|
Average Yield: HeLa Cells (1 x 106 cells) HeLa Cells (1 x 106 cells) HeLa Cells (1 x 106 cells) |
15 μg RNA 8 μg DNA 150 μg protein |
Storage Conditions and Product Stability
The Protein Loading Dye should be stored at -20°C after the addition of DL-Dithiothreitol (DTT). All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 1 year after the date of shipment.
| Component | Cat. 47700 (50 preps) | Cat. 51600 (50 preps) | Cat. 51700 (96 preps) |
|---|---|---|---|
| Buffer SKP | 40 mL | 40 mL | - |
| Lysis Buffer Q | - | - | 40 mL |
| Wash Solution A | 2 x 38 mL 1 x 18 mL |
2 x 38 mL 1 x 18 mL |
2 x 38 mL 1 x 18 mL |
| Elution Solution A | 6 mL | 6 mL | 20 mL |
| Elution Buffer F | 15 mL | 15 mL | 2 x 15 mL |
| Wash Solution C | 30 mL | 30 mL | 60 mL |
| Binding Buffer A | 8 mL | 8 mL | 8 mL |
| Elution Buffer C | 8 mL | 8 mL | 30 mL |
| Protein Neutralizer | 4 mL | 4 mL | 4 mL |
| Protein Loading Dye | 2 mL | 2 mL | 3 x 2 mL |
| gDNA Purification Columns | 50 | - | - |
| gDNA Purification Micro Columns | - | 50 | - |
| gDNA Purification 96-Well Plate | - | - | 1 |
| RNA/Protein Purification Columns | 50 | - | - |
| RNA/Protein Purification Micro Columns | - | 50 | - |
| RNA/Protein Purification 96-Well Plate | - | - | 1 |
| Collection Tubes | 150 | 150 | - |
| Collection Plate | - | - | 5 |
| Elution Tubes (1.7 mL) | 150 | 150 | - |
| Elution Plate | - | - | 3 |
| Lysis Preparation Plate | - | - | 2 |
| Adhesive Tape | - | - | 4 |
| Product Insert | 1 | 1 | 1 |
Documentation
Sequential Purification of RNA, DNA and Protein from a Single Sample using Norgen's RNA/DNA/Protein Purification Kit and Comparison to a Market Competitor
Supplementary Protocol for Total RNA Isolation from Staphylococcus aureus-Related Bacteria and Hard-to-Lyse Bacteria Species
FAQs
Plus, Micro Plus, High Throughput Plus
Poor DNA recovery may be caused by one or more of the following:
- Incomplete lysis of cells or tissue. Ensure that the appropriate amount of Buffer SKP was used for the amount of cells or tissue.
- Columns/wells have become clogged. Do not exceed the recommended amounts of starting materials. The amount of starting material may need to be decreased if the column shows clogging below the recommended levels. See FAQ related to “Clogged Columns/wells” below.
- An alternative elution solution was used. It is recommended that the RNA Elution Buffer supplied with this kit be used for maximum RNA recovery.
- Ethanol was not added to the lysate. Ensure that the appropriate amount of ethanol is added to the lysate before binding to the column/wells.
- Ethanol was not added to the Wash Solution A. Ensure that 96 – 100% ethanol is added to the supplied Wash Solution prior to use.
- Low RNA content in cells or tissues used. Different tissues and cells have different RNA contents, and thus the expected yield of RNA will vary greatly from these different sources. Please check literature to determine the expected RNA content of your starting material.
- Cell Culture: Cell monolayer was not washed with PBS. Ensure that the cell monolayer is washed with the appropriate amount of PBS in order to remove residual media from cells.
- Yeast: Lyticase was not added to the Resuspension Buffer. Ensure that the appropriate amount of lyticase is added when making the Resuspension Buffer.
- Bacteria and Yeast: All traces of media not removed. Ensure that all media is removed prior to the addition of the Buffer SKP through aspiration.
Column/well clogging may occur due to:
- Insufficient solubilization of cells or tissues. Ensure that the appropriate amount of lysis buffer was used for the amount of cells or tissue.
- Maximum number of cells or amount of tissue exceeds kit specifications. Refer to specifications to determine if the amount of starting material falls within kit specifications.
- Centrifuge temperature is too low. Ensure that the centrifuge remains at room temperature throughout the procedure. Temperatures below 20°C may cause precipitates to form that can cause the columns to clog.
RNA may be degraded due to:
- RNase contamination. RNases may be introduced during the use of the kit. Ensure proper procedures are followed when working with RNA. Please refer to “Working with RNA” at the beginning of this user guide.
- Procedure not performed quickly enough. In order to maintain the integrity of the RNA, it is important that the procedure be performed quickly. This is especially important for the Cell Lysate Preparation Step in the Animal Tissue protocol, since the RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized.
- Improper storage of the purified RNA. For short term storage, RNA samples may be stored at –20°C for a few days. It is recommended that samples be stored at –70°C for longer term storage.
- DNase I used may not be RNase-free. Ensure that the optional DNase I being used with this kit is RNase-free in order to prevent possible problems with RNA degradation. Norgen’s RNase-Free DNase I Kit (Cat# 25710) is recommended for this step.
- Lysozyme or lyticase used may not be RNase-free. Ensure that the lysozyme and lyticase being used with this kit is RNase-free, in order to prevent possible problems with RNA degradation.
- Frozen tissues or cell pellets were allowed to thaw prior to RNA isolation. Samples should be flash-frozen in liquid nitrogen and transferred immediately to a -70°C freezer for long-term storage.
If the RNA does not perform well in downstream applications, it may be due to one or more of the following:
- RNA was not washed twice with the provided Wash Solution A. Traces of salt from the binding step may remain in the sample if the columns/wells are not washed twice with Wash Solution A. Salt may interfere with downstream applications, and thus must be washed from the columns/wells.
- Ethanol carryover. Ensure that the dry spin under the RNA Wash procedure is performed, in order to remove traces of ethanol prior to elution. Ethanol is known to interfere with many downstream applications.
A low genomic DNA yield may be caused by:
- Incomplete lysis of cells or tissue. Ensure that the appropriate amount of Buffer SKP was used for the amount of cells or tissue. Incubate the lysate for an extra 10 minutes at 55°C to assist in lysis.
- The DNA elution is incomplete. Ensure that centrifugation at 14,000 x g for 1 minute is performed following the 2 minute centrifugation at 200 x g. Also, ensure that the entire volume of Elution Buffer F passed through and is eluted from the columns/wells.
Sheared genomic DNA could be caused by:
- Sample is old. Ensure that the sample is not too old, as old samples often yield only degraded DNA.
- Sample repeatedly frozen and thawed. Samples should not be repeatedly frozen and thawed, as this tends to increase the likelihood of isolating degraded DNA.
The contamination of RNA with genomic DNA may be because the number of cells or amount of tissue used is close to the maximum recommended amount. When the maximum recommended amount of cells or tissues is used for the procedure, some cross contamination of genomic DNA in the RNA fraction may be observed. Reduce the input amount of cells or tissues below the maximum recommendation in order to avoid this problem. Recommended amounts of starting material to use for optimal kit performance are given in each section of the protocol. Alternatively, carry out the on-column DNase I treatment as described in Appendix B.
Poor protein recovery may be caused by one or more of the following:
- Incorrect pH adjustment of sample. Ensure that the pH of the starting protein sample is adjusted to pH 3.5 or lower after the Binding Buffer A has been added and prior to binding to the columns/wells. If necessary, add additional Binding Buffer A.
- Low protein content in the starting materials. Run a 20 µL fraction from the flowthrough (after RNA binding) on an SDS-PAGE gel to estimate the amount of protein present in the sample. In addition, use the entire flowthrough in the protein purification procedure.
Protein may be degraded due to:
- Eluted protein solution was not neutralized. Add 9.3 µL of Protein Neutralizer to each 100 µL of eluted protein to adjust the pH to neutral. Some proteins are sensitive to high pH, such as the elution buffer at pH 12.5.
- Eluted protein was not neutralized quickly enough. If eluted proteins are not used immediately, degradation will occur. We strongly suggest adding Protein Neutralizer to lower the pH promptly.
- Eluted protein was not stored properly. Purified protein needs to be stored at -70°C. Adding protease inhibitors will help enhance protein stability.
Yes, this kit is able to isolate the Episomal DNA as well. Please contact our Tech support team at support@norgenbiotek.com and ask for reference publications.
Yes, the eluted protein and elution buffer C (+ protein neutralizer) are compatible with applications like Mass Spectrometry.
