NORGEN - Biotech Corporation
Norgen’s Urine DNA Isolation Maxi Kit (Slurry Format) provides a fast, reliable and simple procedure for isolating DNA from 25 mL of urine. DNA found in urine can be divided into 2 basic categories. The larger species (genomic-DNA) is generally greater than 1 kb in size, and appears to be derived mainly from cells shed into the urine. The second species is smaller, generally between 150 and 250 bp (apoptotic-DNA), and derives, at least in part, from the circulation. The second species is also considered as an RNA/DNA hybrid as reported by Halicka et al., 2000. Both types of DNA can be isolated reliably using this kit. Typical yields of DNA isolated will vary depending on the input sample, with more concentrated samples tending to yield more DNA. Preparation time for a single sample is less than 30 minutes. The purified urine DNA is compatible with PCR and Southern Blot analysis.
This kit is designed to process 50 urine samples with a volume of 25 mL urine each
Kit Components:
Customer-Supplied Reagents and Equipment
• Centrifuge with a swinging bucket rotor capable of 2000 x g
• Benchtop microcentrifuge
• Micropipettors
• 96 – 100% ethanol
• 60°C incubator
• 25 mL tubes
Storage Conditions and Product Stability
All buffers should be kept tightly sealed and stored at room temperature (15-25oC). These reagents can be stored for up to 1 year without showing any reduction in performance.
Norgen’s Urine DNA Isolation Maxi Kit (Slurry Format) contains ready-to-use Proteinase K and Pronase solution, which are dissolved in a specially prepared storage buffer. The Proteinase K and the Pronase are stable for up to 1 year after delivery when stored at room temperature. To prolong the lifetime of Proteinase
K and Pronase, storage at 2–8°C is recommended.
Quality Control
In accordance with Norgen’s Quality Management System, each lot of Norgen’s Urine DNA Isolation Maxi Kit (Slurry Format) is tested against predetermined specifications to ensure consistent product quality.
Product Use Limitations
Norgen’s Urine DNA Isolation Maxi Kit (Slurry Format) is designed for research purposes only. It is not intended for human or diagnostic use
Product Warranty and Satisfaction Guarantee
NORGEN BIOTEK CORPORATION guarantees the performance of all products in the manner described in our product manual. The customer must determine the suitability of the product for its particular use.
Safety Information
Ensure that a suitable lab coat, disposable gloves and protective goggles are worn when working with chemicals.
The Binding Solution I, Binding Solution II, Wash Solution I and Wash Solution II contain guanidine hydrochloride, and should be handled with care. Guanidine hydrochloride forms highly reactive compounds when combined with bleach, thus care must be taken to properly dispose of any of these solutions.
If liquid containing these buffers is spilt, clean with suitable laboratory detergent and water. If the spilt liquid contains potentially infectious agents, clean the affected area first with laboratory detergent and water, and then with 1% (v/v) sodium hypochlorite.
Procedure
Notes prior to use:
• We recommend the use of Norgen’s Urine Preservative when collecting urine samples, which is designed for the preservation of nucleic acids and proteins in fresh urine samples at ambient temperatures. The components of the Urine Preservative allow samples to be stored for over 2 years at room temperature with no detected degradation of urine DNA, RNA or proteins. Norgen’s Urine Preservative is available in 2 convenient formats: in a liquid format in Norgen’s Urine Preservative Single Dose Ampules, as well as in a dried format in Norgen’s Urine Collection and Preservation Tubes. Please see the Related Products table below.
• Do not spin down or filter the urine sample before proceeding with the isolation, as this could decrease the DNA yield.
• Ensure that all solutions are at room temperature prior to use, and that no precipitates have formed. If necessary, warm the solutions and mix well until the solutions become clear again.
• Always vortex both the Proteinase K and the Pronase before use.
• Prepare a working concentration of Binding Solution II and Wash Solution I by adding the proper volume of 96-100% ethanol indicated in Table 1 below (provided by the user) to the supplied bottle containing the concentrated Binding Solution II and Wash Solution I. The label on the bottle has a box that may be checked to indicate that the ethanol has been added.
• Preheat an incubator or heating block to 60°C.
• Binding Solution I contains resin, and must be mixed well before each pipetting
Table 1. Volume of Ethanol to be added to Binding Solution II and Wash Solution I
Detailed Procedure
1. Add 3.5 mL of Binding Solution I to every 25 mL urine sample, mix well by inversion; (Note: Binding Solution I contains resin and must be mixed well before every pipeting)
2. Centrifuge for 5 minute at 2,000 x g, then discard the supernatant;
3. Add 100 μL of both Proteinase K and Pronase to the precipitated slurry pellet resulting from 25 mL of urine sample; Vortex for 10 seconds;
4. Incubate the mixture at 60°C for 20 minutes;
5. After the 20 minute incubation, add 260 μL Binding Solution II, mix well by pipeting and then transfer the entire contents into a Mini Filter Spin column;
6. Centrifuge for 2 minutes at 6,700 x g, discard the flow-through;
7. Apply 400 μL of Wash Solution I to the column and centrifuge for 1 minute. Discard the flowthrough and reassemble the spin column with its collection tube;
8. Apply 400 μL of Wash Solution II to the column and centrifuge for 1 minute. Discard the flow-through and reassemble the spin column with its collection tube;
9. Apply 400 μL of 96-100% ethanol (supplied by the user) to the column and centrifuge for 1 minute at 14,000 x g (~14,000 RPM). Discard the flow-through and reassemble the spin column with its collection tube;
10. Repeat Step 9 a second time;
11. Spin the column for 1 minute in order to thoroughly dry the resin. Discard the collection tube;
12. Incubate the column horizontally for 3 minutes at 60°C;
13. Transfer the spin column to a provided Elution tube. Apply 150 μL of Elution Buffer to the column and centrifuge for 2 minutes at 200 x g (~2,000 RPM), followed by 1 minute at 14,000 x g (~14,000 RPM);
14. Transfer the spin column to a fresh 1.7 mL Elution tube. Apply 100 μL of Elution Buffer to the column and centrifuge for 1 minute at 14,000 x g (~14,000 RPM).
Figure 1. A typical 1.2% agarose gel showing total urinary DNA isolated from different urine volumes using Norgen Urine DNA Isolation Kit. Each lane shows one tenth from each elution (i.e. E1: 15 μL out of 150 μL were loaded on the gel, E2: 10 μL out of 100 μL were loaded on the gel ). M: 10 μL Norgen’s FastRunner DNA Ladder.
Frequently Asked Questions
1. If I am not going to process my samples immediately, how should I store my samples?
• We recommend the use of Norgen’s Urine Preservative when collecting urine samples, which is designed for the preservation of nucleic acids and proteins in fresh urine samples at ambient temperatures. Urine samples in the preservative should be stored at room temperature. Turbidity or precipitation may be observed if the urine samples are stored at either 4°C or at -20°C. DO NOT discard this precipitate and/or spin down your samples to get rid of the turbidity; this will significantly reduce your DNA yields. Make sure to mix your samples thoroughly before processing.
2. What If a variable speed centrifuge is not available?
• If a variable speed centrifuge is not available a fixed speed centrifuge can be used, however reduced yields may be observed.
3. What will happen if my centrifugation speed varied from the recommended centrifugation speed?
• This may lead to the degradation of the genomic DNA or reduction in the total DNA yields may be observed.
4. At what temperature should I centrifuge my samples?
• All centrifugation steps are performed at room temperature. Centrifugation at 4°C will not adversely affect kit performance.
5. My centrifuge speeds are defined in rpm and not in g-force, how can I convert g-force to rpm?
• The correct rpm can be calculated using the formula:
where RCF = required gravitational acceleration (relative centrifugal force in units of g); r = radius of the rotor in cm; and RPM = the number of revolutions per minute required to achieve the necessary g-force.
6. What If I added more or less from the specified reagents’ volume?
• Adding less volume may reduce your DNA yields. Adding more may not affect the DNA yields EXCEPT if more Elution Buffer was added. Eluting your DNA in a higher volume of Elution Buffer will result in diluting your DNA.
7. What If my incubation temperature varied from the specified 60°C?
• The incubation temperature can be in the range of 55°C - 65°C. Outside of this range the activity of both the Proteinase K and the Pronase will be reduced. This will result in a reduction in your DNA yields.
8. What If my incubation varied from the 20 minutes specified in the product manual?
• Less than 20 minutes will result in a lower DNA yields. More than 20 minutes may not affect your DNA yields.
9. What If I forgot to do a dry spin after my second wash?
• Your first DNA elution will be contaminated with the wash solutions. This may dilute the DNA yield in your first elution. Also, it may interfere with your downstream applications.
10. Can I have a third elution?
• Yes, you can. A third elution is possible but in a smaller volume (50 μL)
11. Why am I eluting my DNA into two elutions?
• The first elution will mainly contain the low nucleic acid species and traces from the large DNA species. However, your second elution will mainly contain the large DNA species and traces from the low nucleic acid species (Please see Figure 1).
12. Why my samples showed very low DNA yield?
• Some urine samples contain very little DNA. This varies from individual to individual based on numerous variables. In order to increase the yield, the amount of urine input could be increased. Increasing the incubation time at 65ºC (up to overnight) could also result in increased yields.
13. Why my DNA does not perform well in downstream applications?
• If a different Elution Buffer was used other than the one provided in the kit, the buffer should be checked for any components that may interfere with the application. Common components that are known to interfere are high salts (including EDTA), detergents and other denaturants. Check the compatibility of your elution buffer with the intended use.
14. What is the expected DNA yield from urine?
• The urinary DNA yield varies between individual samples. The DNA yield ranges between 50 ng – 2 μg/mL of urine sample. In many cases DNA yields from urine are too low to be visualized on an agarose gel, however the DNA yield is sufficient for most of the downstream applications including PCR and Southern hybridization.
15. I am noticing a white precipitate in my elution. What should I do?
• This white precipitate may appear in the elution depending on the nature of the sample. This white precipitate will not affect your downstream application. Simply mix your elution well before using.
Reference
Halicka, H. D., Bedner, E. and Darzynkiewicz, Z. (2000). Segregation of RNA and separate packaging of
DNA and RNA in apoptotic bodies during apoptosis. Exp Cell Res. 260, 248-256.
M. Abdalla and Y. Haj-Ahmad. (2006).Urinary Proteomic and Genomic Profiles from Hepatitis C virus,
Hepatitis B Virus, and Hepatocellular Carcinoma Patients. Molecular & Cellular Proteomics. ASBMB, S369