Navigation Links
M13/Phagemid DNA Extraction Protocol

Efficiency of the standard protocols(1,2) for production of single-stranded DNA may be improved through the use of PLG.(3)
  1. Propagate M13 phage by one of the standard procedures:
    1. For producing M13 ssDNA, mix 2.5 ml plating bacteria (57 hours, 50 ml culture; stored at 4 C for no more than 3 days) with 300 l M13 phage stock (1 x 1011 pfu/ml) and incubate 5 minutes at room temperature to allow phage to interact with the bacterial phage receptors. The phage-bacteria culture is added to 250 ml LB broth and incubated for 57 hours at 37C with sufficient agitation for good aeration of the culture.
    2. For producing phagemid ssDNA, suspend a colony of phagemid transformed bacteria in a 15 ml culture tube containing 3 ml LB broth. Helper phage M13K07 is added to a final concentration of 2 x 107 pfu/ml and the culture is incubated for 1.5 hours at 37C with sufficient agitation. Recover resultant supernatant to a fresh tube or bottle and re-centrifuge as above.
  2. Recover resultant supernatant to a fresh centrifuge tube, add 0.25 volume of 20% PEG 8000/2.5 M NaCl to the supernatant, mix thoroughly by repeated inversion, and incubate 20 minutes at room temperature. Centrifuge at 15,500 x g, 4C, for 20 minutes and discard the resultant supernatant. Allow any traces of supernatant to drain from the tube.
  3. Resuspend pellet in 50 l TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) for every 1 ml of supernatant (step 3) and transfer the entire sample to a pre-spun (1500 x g fo r 12 minutes) PLG 15 ml Light tube.
  4. Add an equal volume of Tris-buffer saturated Phenol (pH 8.0) to the sample in the PLG 15 ml tube, and mix thoroughly by repeated inversion. Centrifuge at 1500 x g for 5 minutes to separate the phases.
  5. Add an equal volume of Phenol-Chloroforrn-Isoamyl Alcohol (PCI, 25:24:1) to the sample in the same PLG 15 ml tube and extract and centrifuge as in step 5.
  6. Add an equal volume of Chloroform-Isoamyl Alcohol (CI, 24:1) to the sample in the same PLG 15 ml tube and extract and centrifuge as in step 4.
  7. Quantitatively transfer final aqueous phase to a 2 mL microcentrifuge tube, add 3 volumes of a 25:1 mixture of absolute Ethanol:3 M Sodium Acetate (pH 5.2), and mix well.
  8. Let stand for 1530 minutes at room temperature, and centrifuge for 20 minutes at 12,000 x g or greater at 4C.
  9. Wash pellet twice with 0.5 ml 70% Ethanol and air-dry.
  10. Resuspend pellet in 2050 l TE, pH 8.0.
  1. Current Protocols in Molecular Biology, Volume I. (1989) Ausubel, F.M. et al. (eds.). John Wiley and Sons, New York, NY pp.1.14.1 - 1.15.4.
  2. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. pp.4.29-4.32.
  3. PLG Notes. (1992) Prime Report 4(2):



Page: All 1 2

Related biology technology :

1. M13/Phagemid DNA Extraction Protocol
2. Lambda DNA Extraction Protocol
3. Genomic DNA Extraction from Buccal Swabs Using the Perfect gDNA Blood Mini Kit
4. Genomic DNA Extraction from Buffy Coat Using the Perfect gDNA Blood Mini Kit
5. Lambda DNA Extraction Protocol
6. Genomic DNA Extraction from Buccal Swabs Using the Perfect gDNA Blood Mini Kit
7. Genomic DNA Extraction from Buffy Coat Using the Perfect gDNA Blood Mini Kit
8. S.E.T.S. Swab Extraction Tube System
9. InstaGene Matrix: Prepare DNA Templates for PCR With No Phenol/Chloroform Extractions and No Deproteinization Steps, Rev C
10. New Protocols for Isolating High- Molecular-Weight Genomic DNA
11. Eppendorf Multiporator Transfection Protocols for Eukaryotic Cells
Post Your Comments:

(Date:6/23/2016)... ... June 23, 2016 , ... Charm Sciences, Inc. is pleased ... received AOAC Research Institute approval 061601. , “This is another AOAC-RI approval of ... Salter, Vice President of Regulatory and Industrial Affairs. “The Peel Plate methods perform ...
(Date:6/23/2016)... , June, 23, 2016  The Biodesign Challenge ... envision new ways to harness living systems and biotechnology, ... Art (MoMA) in New York City ... 130 participating students, showcased projects at MoMA,s Celeste Bartos ... Paola Antonelli , MoMA,s senior curator of architecture and ...
(Date:6/23/2016)... (PRWEB) , ... June 23, 2016 , ... ... today announced the launch of the Supplyframe Design Lab . Located in ... to explore the future of how hardware projects are designed, built and brought ...
(Date:6/23/2016)... ... ... In a new case report published today in STEM CELLS Translational Medicine, doctors ... being treated for breast cancer benefitted from an injection of stem cells derived from ... frequent side effect of cancer treatment. , Lymphedema refers to the swelling ...
Breaking Biology Technology:
(Date:6/15/2016)... New York , June 15, 2016 /PRNewswire/ ... a new market report titled "Gesture Recognition Market by ... and Forecast, 2016 - 2024". According to the report, ... USD 11.60 billion in 2015 and is estimated ... reach USD 48.56 billion by 2024.  ...
(Date:6/3/2016)... Das DOTM (Department ... hat ein 44 Millionen $-Projekt ... einschließlich Personalisierung, Registrierung und IT-Infrastruktur, an Decatur ... Implementierung von Identitätsmanagementlösungen. Zahlreiche renommierte internationale Anbieter ... aber Decatur wurde als konformste und innovativste ...
(Date:6/1/2016)... YORK , June 1, 2016 ... Technology in Election Administration and Criminal Identification to Boost ... to a recently released TechSci Research report, " Global ... By Region, Competition Forecast and Opportunities, 2011 - 2021", ... 24.8 billion by 2021, on account of growing security ...
Breaking Biology News(10 mins):