Water RNA/DNA Purification Kits

Supporting Data

Figure 1. High Yield and Purity of RNA and DNA. Total RNA and DNA were simultaneously isolated from 50 mL of water sample containing 10⁷cfu/mL E.coli using Norgen's Water RNA/DNA Purification Kit and subsequently run on gels for visual analysis. Panel A shows 10 µL aliquots (no RNase treatment) of the 50 µL elutions run on a 1% TAE agarose gel. Genomic DNA and 16S and 23S rRNA bands were visible. Panel B shows 5 µL aliquots (on-column DNase was applied) of the elution run on a 1.5% formaldehyde agarose gel. 16S and 23S rRNA was seen without DNA contamination. From observing the gels it can be seen that the kit allows for the isolation and purification of high yields of concentrated and high quality RNA and DNA.

Figure 2. Detection of E. coli 16S rDNA by Real-Time PCR (SYBR Green) Fifty mL water samples spiked with increasing amounts of E.coli were filtrated through the provided 0.45 µm water filter columns, and RNase-treated DNA was isolated from the filter by following Norgen's Water RNA/DNA Purification Kit manual. Next, 2 µL of DNA from the 50 µL elutions was mixed in 18 µL of total real-time PCR reaction master mix and real-time PCR was performed (95°C for 3 minutes and 40 cycles at 95°C for 15 second and 60°C for 30 seconds.). All tested water samples containing different E.coli amounts were found to correspond to the real-time PCR results. E.coli was specifically detected by E. coli 16S rDNA primers down to 10² cfu/ml, indicating the quality of DNA and the efficiency of filtering system.

Kit Specifications
Minimum Water Input	10 mL
Maximum Water Input	100 mL
Maximum Filter Column Loading Volume	20 mL
Maximum Spin Column Loading Volume	650 µL
Elution Volume	100 µL
Time to Complete 10 Purifications	45 minutes

Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years from the date of shipment.

Component	Cat. 26400 (25 preps)	Cat. 26450 (25 preps)	Cat. 26480 (50 preps)
Lysis Buffer E	15 mL	15 mL	2 x 15 mL
Wash Solution A	18 mL	18 mL	38 mL
Enzyme Incubation Buffer B	6 mL	6 mL	6 mL
Elution Buffer H	6 mL	6 mL	6 mL
Mini Spin Columns	25	25	50
Filter Columns (0.22 μL)	25	-	-
Filter Columns (0.45 μL)	-	25	-
Bead Tubes	25	25	50
Collection Tubes	25	25	50
Elution Tubes (1.7 mL)	25	25	50
Product Insert	1	1	1

Documentation

PROTOCOLS

(26400) Water RNA/DNA Purification Kit (Spin Column + 0.22 µm Filter) - Protocol (0.22 µm)

(26450) Water RNA/DNA Purification Kit (Spin Column + 0.45 µm Filter) - Protocol (0.45 µm)

(26480) Water RNA/DNA Purification Kit (Spin Column) - Protocol (50 preps)

SAFETY DATA SHEETS

26400 - Water RNA_DNA Purification Kit (Spin Column + 0.22 um Filter) - SDS

26450 - Water RNA_DNA Purification Kit (Spin Column + 0.45 um Filter) - SDS

26480 - Water RNA_DNA Purification Kit (Spin Column) - SDS

FAQs

Spin Column + 0.22 µm Filter, Spin Column + 0.45 µm Filter, Spin Column

I noticed the column was clogged. What causes this?

Column clogging can result from one or a combination of the following factors:

Input volume of water sample was too high.
The amount of sample input may need to be decreased to prevent clogging of the Filter Column, particularly for turbid water samples.
Pre-filtering is necessary.
For highly turbid water samples, or samples containing high levels of sediments, pre-filtration of the sample might be necessary. Pass the sample through a 1-8 µm filter to remove debris prior to applying the sample to the Filter Column.

Why do I have poor DNA/ RNA recovery?

Lysis was not completed.

Ensure the filter has not dried out during the 65℃ incubation step. Alternatively, increase the incubation time at 65℃ to 15 minutes.
Ethanol was not added to the lysate.
Ensure that the appropriate amount of ethanol is added to the lysate before binding to the column.
Ethanol was not added to the Wash Solution.
Ensure that 42 mL of 96-100% ethanol is added to the supplied Wash Solution A prior to use.
An alternative elution buffer was used.
It is recommended that the Elution Buffer H supplied with this kit be used for maximum DNA recovery.

Why is the DNA/RNA not performing well in downstream applications?

If the DNA or RNA does not perform well in downstream applications, it may be due to one or more of the following:

Ethanol carryover.
Ensure that the dry spin under the Column Wash procedure is performed in order to remove traces of ethanol prior to elution. Ethanol is known to interfere with many downstream applications.
DNA/RNA was not washed twice with the provided Wash Solutions A.
Traces of salt from the binding step may remain in the sample if the column is not washed twice with the Wash Solution A. Salt may interfere with downstream applications and thus must be washed from the column.
PCR reaction conditions need to be optimized.
Take steps to optimize the PCR conditions being used, including varying the amount of template, changing the source of Taq polymerase, looking into the primer design, and adjusting the annealing conditions.