Phage DNA Isolation Kit

Features and Benefits

Isolate high quality DNA from a broad variety of phage strains
High yields of total DNA
Fast and easy processing using a rapid spin-column format
No phenol or chloroform extractions or cesium chloride banding required
High yields of DNA recovered 3-15 µg DNA from 10⁸-10¹⁰pfu/ mL of enriched phages

This kit provides a rapid spin column method for the purification of total DNA from a broad spectrum of bacteriophages propagated in bacteria grown in liquid cultures. The DNA is isolated without the use of phenol, chloroform or cesium chloride banding procedures. The spin-column based procedure is rapid and can be completed in less than 45 minutes. The kit is highly efficient for processing small volumes of phage supernatant (500 µL – 1 mL) and with the optional DNase and Proteinase K treatments phage DNA yields are maximized while host DNA contamination is minimized. Purified total phage DNA is of the highest integrity, and can be used in a number of downstream applications including PCR, qPCR, Restriction Fragment Length Polymorphism (RFLP), sequencing, cloning, Southern Blot and more.

Details

Supporting Data

Figure 1. Effective Host Genomic DNA Removal without Reducing Phage DNA Yield. Total DNA was isolated from four enriched phage cultures using Norgen's Phage DNA Isolation Kit. A DNase I pre-treatment was performed prior to adding the provided Lysis Buffer. Briefly, 20 units of DNase I was added to 1 mL of enriched phage culture and the mixture was incubated at room temperature for 20 minutes. After the DNAase I treatment the procedure was followed. As a control, DNA was isolated from aliquots of the same 4 cultures using Norgen’s Phage DNA Isolation Kit without performing the DNase I treatment. For DNA analysis 10 µL of each 50 µL elution was loaded onto a 1X TAE agarose gel. As it can be seen, the phage DNA was safely protected from the DNase I treatment by its coat protein, while the host genomic DNA was efficiently degraded by the DNase I. Thus the DNase I pre-treatment resulted in less host gDNA contamination in the final phage elution without influencing the total phage DNA yield. Lane M is Norgen's Highranger 1 kb DNA Ladder (Cat. 11900)

Figure 2. Optional Proteinase K Treatment Improves DNA Yield for Certain Phage Strains. Total DNA was isolated with and without the optional Proteinase K treatment using Norgen's Phage DNA Isolation Kit. Briefly, 4 µL of Proteinase K (20 mg/mL) was added to 1 mL of enriched phage culture and incubated at 55°C for 15 minutes with the phage Lysis Buffer. After the Proteinase K treatment the procedure was followed. As a control, DNA was isolated from aliquots of the same 8 cultures using Norgen's Phage DNA Isolation Kit without performing the Proteinase K treatment. For DNA analysis 10 µL of each 50 µL elution was loaded onto a 1X TAE agarose gel and the yield of DNA was compared from the eight different phage types (lane 1 to 8). As it can be seen, the optional treatment of Proteinase K improved the phage DNA yield in Lanes 2, 5 and 6 dramatically. Lane M is Norgen's Highranger 1 kb DNA Ladder (Cat. 11900)

Kit Specifications
Column Binding Capacity	50 µg
Maximum Column Loading Volume	650 µL
Size of DNA Purified	All sizes
Maximum Amount of Starting Material	1 x 10¹⁰ pfu/mL enriched phages
Average Yield*	3-15 µg DNA from 10⁸-10¹⁰ pfu/mL of enriched phages
Time to Complete 10 Purifications	45 minutes

* Average yields will vary depending upon a number conditions used and developmental stage.

Storage Conditions and Product Stability
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. 46800 (50 preps)	Cat. 46850 (100 preps)
Lysis Buffer B	40 mL	2 x 40 mL
Wash Solution A	38 mL	2 x 38 mL
Elution Buffer B	8 mL	2 x 8 mL
Spin Columns	50	100
Collection Tubes	50	100
Elution Tubes (1.7 mL)	50	100
Product Insert	1	1

Documentation

FAQs

Spin Column

I noticed the column was clogged. What causes this?

Column clogging may occur due to one or more of the following reasons:

Centrifugation speed was too low or spin time was inadequate.
Check the centrifuge to ensure that it is capable of generating the required RPMs. Sufficient centrifugal force is required to move the liquid through the resin. Also, ensure that the correct spin times are followed. Spin for an additional minute if necessary.
Bacterial debris in the lysate.
Ensure that the starting material is clarified phage supernatant. Remove bacterial debris from the initial phage supernatant by centrifugation at 10,000 × g for 5 minutes before beginning the protocol.
The lysate/binding solution mixture is not homogeneous.
To ensure a homogeneous solution, vortex for 10-15 seconds before applying the lysate to the spin column.
Centrifuge temperature is too low.
Ensure that the centrifuge remains at room temperature throughout the procedure. Temperatures below 20℃ may cause precipitates to form that can cause the columns to clog.

Why do I have low yield of phage DNA?

The yield of genomic DNA may be lower than expected due to the following:

Ineffective propagation of phage and initial lysis step.
Refer to the manufacturer's recommendations for the propagation of the phage, including proper titer for inoculation, growth conditions, and bacterial host.
Incomplete lysis of cells.
Ensure that the incubation was performed for 15 minutes at 65℃ after the addition of Lysis Buffer B. Also, perform optional digestion with Proteinase K in Step 1b during Lysate Preparation.
Ethanol was not added to the Wash Solution A.
Ensure that 90 mL of 96 - 100% ethanol is added to the supplied Wash Solution A prior to use.
The DNA elution is incomplete.
Ensure that all the DNA is eluted. If elution buffer remains in the column, use 14,000 g for the second centrifuge.

Why is the purified DNA not performing well in downstream applications?

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

DNA was not washed three times with the provided Wash Solution A.
Traces of salt from the binding step may remain in the sample if the column is not washed three times with the Wash Solution A. Salt may interfere with downstream applications and thus must be washed from the column.
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.

Why do I have host genomic DNA contamination in phage DNA elution?

Host genomic DNA can contaminate the phage DNA. In order to eliminate host genomic DNA contamination in the phage DNA elution, it is recommended that a DNase I treatment is performed at the beginning of the lysate preparation procedure (see Section 1, Optional DNase Treatment).

Can this kit isolate phage genomes with ssDNA ?

Yes, the kit can isolate both ssDNA and dsDNA phage genomes.

Can PEG precipitation be used before the Phage DNA isolation kit?

The kit doesn't require PEG precipitation. However, the kit is compatible with PEG precipitated samples.

Citations

Title	Bacteriophages with potential to inactivate Salmonella Typhimurium: use of single phage suspensions and phage cocktails
Citation	Virus Research 2016.
Authors	Pereira, C., Moreirinha, C., Lewicka, M., Almeida, P., Clemente, C., Cunha, Â., ... & Almeida, A

Title	Identification of essential genes in the Salmonella phage SPN3US reveals novel insights into giant phage head structure and assembly.
Citation	Journal of Virology 2016.
Authors	Thomas, J. A., Quintana, A. D. B., Bosch, M. A., De Peña, A. C., Aguilera, E., Coulibaly, A., ... & Black, L. W

Title	Characterization and in vitro evaluation of new bacteriophages for the biocontrol of Escherichia coli
Citation	Virus Research 2016.
Authors	Pereira, C., Moreirinha, C., Lewicka, M., Almeida, P., Clemente, C., Romalde, J. L., ... & Almeida, A

Title	Characterization of Five Novel Brevibacillus Bacteriophages and Genomic Comparison of Brevibacillus Phages
Citation	PLoS One 2016.
Authors	Berg, J. A., Merrill, B. D., Crockett, J. T., Esplin, K. P., Evans, M. R., Heaton, K. E., ... & Simister, A. R

Title	The presence of two receptor-binding proteins contributes to the wide host range of staphylococcal Twort-like phages
Citation	Applied and Environmental Microbiology 2016.
Authors	Takeuchi, I., Osada, K., Azam, A. H., Asakawa, H., Miyanaga, K., & Tanji, Y

Title	The diversity-generating benefits of a prokaryotic adaptive immune system
Citation	Nature 2016.
Authors	van Houte, S., Ekroth, A. K., Broniewski, J. M., Chabas, H., Ashby, B., Bondy-Denomy, J., ... & Westra, E. R

Title	The complete genome sequence and analysis of vB_VorS-PVo5, a Vibrio phage infectious to the pathogenic bacterium Vibrio ordalii ATCC-33509
Citation	Standards in Genomic Sciences 2016.
Authors	Echeverría-Vega, A., Morales-Vicencio, P., Saez-Saavedra, C., Ceh, J., & Araya, R

Title	A virulent phage JHP against Pseudomonas aeruginosa showed infectivity against multiple genera
Citation	Journal of Basic Microbiology 2016.
Authors	Khawaja, K. A., Rauf, M., & Abbas, Z

Title	Acquisition of Phage Sensitivity by Bacteria through Exchange of Phage Receptors
Citation	Cell 2016.
Authors	Tzipilevich, E., Habusha, M., & Ben-Yehuda, S

Title	Development of an Assay for the Identification of Receptor Binding Proteins from Bacteriophages
Citation	Viruses 2016.
Authors	David J. Simpson, Jessica C. Sacher and Christine M. Szymanski