Phage DNA Isolation Kit
For the rapid purification of total DNA from bacteriophages.
For research use only and NOT intended for in vitro diagnostics.
Phage DNA Isolation Kit
For the rapid purification of total DNA from bacteriophages.
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 108-1010 pfu/ 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
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
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.
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.
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.
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).
Yes, the kit can isolate both ssDNA and dsDNA phage genomes.
Citations
Title | Phage Encoded Colanic Acid-Degrading Enzyme Permits Lytic Phage Infection of a Capsule-Forming Resistant Mutant Escherchia coli Strain |
Citation | Applied and Environmental Microbiology 2023. |
Authors | MS Kim, YD Kim, SS Hong, K Park, KS Ko, H Myung |
Title | Phage Paride can kill dormant, antibiotic-tolerant cells of Pseudomonas aeruginosa by direct lytic replication |
Citation | Nature Communications 2023. |
Authors | Enea Maffei, Anne-Kathrin Woischnig, Marco R. Burkolter, Dorentina Humolli, Nicole Thürkauf, Thomas Bock, Alexander Schmidt, Pablo Manfredi, Adrian Egli, Nina Khanna, Urs Jenal & Alexander Harms |
Title | Phage-antibiotic synergy reduces Burkholderia cenocepacia population |
Citation | BMC Microbiology 2023. |
Authors | Anna G. Mankovich, Kristen Maciel, Madison Kavanaugh, Erin Kistler, Emily Muckle & Christine L. Weingart |
Title | Phenotypic and Genotypic Characterization of Newly Isolated Xanthomonas euvesicatoria-Specific Bacteriophages and Evaluation of Their Biocontrol Potential |
Citation | Plants 2023. |
Authors | Toana Kizheva, Zoltan Urshev, Melani Dimitrova, Nevena Bogatzevska, Penka Moncheva and Petya Hristova |
Title | Propionibacterium acnes Bacteriophages Display Limited Genetic Diversity and Broad Killing Activity against Bacterial Skin Isolates |
Citation | mBio 2023. |
Authors | Laura Marinelli, Sorel Fitz-Gibbon, Clarmyra Hayes, Charles Bowman, Megan Inkeles, Anya Loncaric, Daniel Russell, Deborah Jacobs-Sera, Shawn Cokus, MatteoPellegrini, Jenny Kim, Jeff Miller, Graham Hatfull, Robert Modlin |
Title | Protocol for phage matching, treatment, and monitoring for compassionate bacteriophage use in non-resolving infections |
Citation | STAR Protocols 2023. |
Authors | Hadil Onallah 1 2 3 6, Ortal Yerushalmy 2 4 6, Ron Braunstein 2 4, Sivan Alkalay-Oren 2 4, Amit Rimon 2 4 5, Daniel Gelman 2 4 5, Shunit Coppenhagen-Glazer 2 4, Ronen Hazan 2 4 7, Ran Nir-Paz 1 2 3 8 |
Title | Recombinant LysVPMS1 as an endolysin with broad lytic activity against Vibrio parahaemolyticus strains associated to acute hepatopancreatic necrosis disease |
Citation | Aquaculture 2023. |
Authors | Zermeño-Cervantes, L. A., Makarov, R., Lomelí-Ortega, C. O., Martínez-Díaz, S. F., & Cardona-Félix, C. S |
Title | Seed coating with phages for sustainable plant biocontrol of plant pathogens 2 and influence of the seed coat mucilage |
Citation | Prepriint BIORXIV 2023. |
Authors | Sebastian H. Erdrich 1,2, Ulrich Schurr 1 , Julia Frunzke 2* and Borjana Arsova 1 |
Title | Sewage as a rich source of phage study against Pseudomonas aeruginosa PAO |
Citation | Biologicals 2023. |
Authors | Reza Aziziana, b, Ahmad Nassera, b, Hasan Askaria, b, Morovat Taheri Kalania, c, Nourkhoda Sadeghifarda, c, Iraj Pakzada, c, Razieh Aminid, Amir Sasan Mozaffari Nejadd, Farid Azizi Jaliliand |
Title | Staphylococcus sciuri bacteriophages double-convert for staphylokinase and phospholipase, mediate interspecies plasmid transduction, and package mecA gene |
Citation | Scientific Reports 2023. |
Authors | Zeman, M., Mašlanová, I., Indráková, A., Šiborová, M., Mikulášek, K., Bendícková, K., ... & Pantucek, R. (2017). |