M13/Phagemid DNA Extraction Protocol ( ...)

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⁾

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.
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.
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
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PCNA Immunohistochemistry Assay Kit from Invitrogen

Varian Cary 6000i Spectrophotometer from Varian, Inc.

Gln(Trt) Peptide Synthesis Column from Applied Biosystems

E. coli Expression System with Gateway Technology from Invitrogen

Biology Products:

PCNA Immunohistochemistry Assay Kit from Invitrogen Varian Cary 6000i Spectrophotometer from Varian, Inc.Gln(Trt) Peptide Synthesis Column from Applied Biosystems E. coli Expression System with Gateway Technology from Invitrogen

( ...)M13/Phagemid DNA Extraction Protocol