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.
References
  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):

'"/>

Source:


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:
*Name:
*Comment:
*Email:


(Date:5/23/2016)... ... May 23, 2016 , ... PrecisionAg® ... Farming in 2017 and Beyond. The paper outlines the key trends that are ... industry. , “We’ve witnessed a lot of highs and lows as the precision ...
(Date:5/23/2016)... CA (PRWEB) , ... May 23, 2016 , ... ... public interest organization focused on molecular nanotechnology, announced the winners for the 2015 ... pioneer physicist Richard Feynman, are given in two categories, one for experiment and ...
(Date:5/20/2016)... Baltimore, MD (PRWEB) , ... May 20, 2016 , ... ... contaminated with Listeria, as reported by Food Safety News on May 12, 2016(1), demonstrates ... according to Ted Olsen, CEO of Baltimore-based biotech firm, PathSensors, Inc. ...
(Date:5/19/2016)... May 19, 2016 ... (OTC PINK: RGBPP) announced today initiation of a ... cord blood based cancer immunotherapeutic product leveraging its ... Regen described a generation of cord blood derived ... gene silencing.  The product in development will be ...
Breaking Biology Technology:
(Date:3/14/2016)... http://www.apimages.com ) - ... - Renvoi : image disponible via AP Images ( ... --> DERMALOG, le leader de l,innovation ... d,empreintes digitales pour l,enregistrement des réfugiés en Allemagne. ... produire des cartes d,identité aux réfugiés. DERMALOG dévoilera ...
(Date:3/10/2016)... March 10, 2016   Unisys Corporation (NYSE: ... Protection (CBP) is testing its biometric identity solution at ... to help identify certain non-U.S. citizens leaving the ... test, designed to help determine the efficiency and accuracy of ... February and will run until May 2016. --> ...
(Date:3/8/2016)... 2016   Valencell , the leading innovator ... has secured $11M in Series D financing. The ... venture fund being launched by UAE-based financial services ... investors TDF Ventures and WSJ Joshua Fund. Valencell ... triple-digit growth and accelerate its pioneering innovation in ...
Breaking Biology News(10 mins):