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 | A gradient phage-assisted continuous evolution method for screening suppressor tRNAs in Escherichia coli |
Citation | New Biotechnology 2023. |
Authors | Fan Wang a b 1, Li-Hua Liu b c 1, Zhenyu Wang a 1, Ao Jiang b 1, Yi-Rui Wu |
Title | A Klebsiella pneumoniae NDM-1+ bacteriophage: Adaptive polyvalence and disruption of heterogenous biofilms |
Citation | Frontiers in Microbiology 2023. |
Authors | Eddie B. Gilcrease, Sherwood R. Casjens, Ananda Bhattacharjee and Ramesh Goel |
Title | A Metagenomic approach to characterize temperate bacteriophage populations from cystic fibrosis and non-cystic fibrosis bronchiectasis patients |
Citation | Frontiers in Microbiology 2023. |
Authors | MA Tariq, FLC Everest, L Cowley, A De Soyza, GS Holt, SH Bridge, A Perry, JD Perry, S Bourke, S Cummings, CV Lanyon, JJ Barr, DL Smith |
Title | A Metagenomics Approach to Enumerate Bacteriophages in a Food Niche |
Citation | Bacteriophages 2023. |
Authors | Kelsey White, Giovanni Eraclio, Gabriele Andrea Lugli, Marco Ventura, Jennifer Mahony, Fabio Dal Bello & Douwe van Sinderen |
Title | A virulent phage JHP against Pseudomonas aeruginosa showed infectivity against multiple genera |
Citation | Journal of Basic Microbiology 2023. |
Authors | Khawaja, K. A., Rauf, M., & Abbas, Z |
Title | Acquisition of Phage Sensitivity by Bacteria through Exchange of Phage Receptors |
Citation | Cell 2023. |
Authors | Tzipilevich, E., Habusha, M., & Ben-Yehuda, S |
Title | AN INNOVATIVE APPROACH USING LYTIC PHAGE MIX FOR WASTEWATER MANAGEMENT AND PATHOGEN CONTROL |
Citation | Egypt Journal of Applied Science 2023. |
Authors | Mohamed I. Azzam 1; Abeer A. Faiesal; Fafy A. Mohammed and A.S. Korayem |
Title | Assessing phage-host population dynamics by reintroducing virulent viruses to synthetic microbiomes |
Citation | Cell Host and Microbe 2023. |
Authors | Jacob Wilde Randy Boyes Avery V. Robinson Brendan A. Daisley Alexander J. Botschner Dylan J.L. Brettingham Christine V. Macpherson Elizabeth Mallory Emma Allen-Vercoe |
Title | Bacteriophage Cocktail Comprising Fifi044 and Fifi318 for Biocontrol of Erwinia Amylovora |
Citation | Plant Pathol J 2023. |
Authors | Byeori Kim, Seung Yeup Lee, Jungkum Park, Sujin Song, Kwang-Pyo Kim, Eunjung Roh |
Title | Bacteriophage vB_kpnS-Kpn15: Unveiling its potential triumph against extended-spectrum beta-lactamase-producing Klebsiella pneumoniae - Unraveling efficacy through innovative animal alternate models |
Citation | Microbial pathogenesis 2023. |
Authors | Eugene Athan |