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Phage-Display Vectors and Libraries Based on Filamentous Phage Strain fd-tet
George P. Smith
Division of Biological Sciences
Tucker Hall
University of Missouri
Columbia, MO 65211-7400
(573) 882-3344 (office)
(573) 882-2720 (lab)
smithgp@ missouri.edu (e-mail)
Distribution Policy
Unless we specifically stipulate otherwise, you are free to pass on any materials obtained from
our lab to any other user. Until further notice, we plan to continue our long-standing policy of
distributing our “kit” (vector and E. coli host strains) and amplified random peptide libraries free
to anyone who asks, including to commercial enterprises. To request materials, download
KitRequest.doc and follow instructions. We no longer accept requests for standard phage-display
materials in other forms, including in the body of e-mails or in letters; such requests will simply
be discarded without notification. However, we do try to respond to other communications,
including requests for special materials not covered in the the KitRequest document.
Standard Preparations and Procedures are referred to in italics
Throughout this manual components that are made from stock solutions given in stdpreps.doc are
printed in italics. Also printed in italics are a number of standard methods that are also
explained in that document.
This Document Contains Descriptions of and Hyperlinks to Other Documents That
Describe Our Phage-Display System and How to Use It
This document contains numerous hyperlinks to other Microsoft Word documents, each of which
details some specific aspect of the phage-display system. In order to be accessible though a
hyperlink, a document must previously have been downloaded from our website at
http://www.biosci.missouri.edu/smithgp/PhageDisplayWebsite/PhageDisplayWebsiteIndex.html.
Clicking on a hyperlink opens the document. The hyperlinks use relative (local) paths, and will
therefore only work if all the documents (including this one) are in the same directory (folder).
The documents are largely self-contained, however, so it isn’t necessary to download all of them.
Whatever else you download (besides the present document), you should certainly download the
above-mentioned stdpreps.doc, which contains standard preparations and procedures referenced
in almost all the other documents.
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List of documents
Contents
Basic review of phage display from Chemical Reviews 1997
Standard preparations (solutions, media, etc.) and
procedures referenced in almost all the other documents
Description and confirmation of E. coli strains
Description of fUSE vectors (display on gene III protein)
and f88 vectors (display on gene VIII protein)
Random peptide libraries in Descriptions of libraries
fUSE5 and f88-4 vectors
Sequences of insert regions
Form for requesting vectors, libraries or strains
Amplifying a large phage-display library without losing
diversity
Biotinylation of a receptor for use in affinity selection and
binding assays
Affinity selection (“biopanning”): using a target selector
(receptor, antibody, other binding molecule) to select
binding clones from large libraries
1-ml scale propagation and processing of clones for
sequencing and binding assays (we never do this any more)
7-ml scale propagation and partial purification of virions
Extraction of ssDNA from virions for sequencing or other
purposes
Manual procedure for sequencing inserts in large numbers
of clones (we never do this any more)
Binding assays
Phage capture assay (miniaturized
for confirming
analytical version of affinity selection,
affinty of phage
in which the input is individual clones
clones for target rather than complex populations)
receptor
Quantifying binding of selector to phage
clones immobilized on the surface of
plastic wells
(Continued next page)
Document
PetrenkoSmithChemReviews.PD
F
stdpreps.doc
Strains.doc
vectors.doc
library.doc
libeseq.doc
KitRequest.doc
AmplifyingLibrary.doc
biotinylation.doc
AffinitySelection.doc
Propagation_1ml.doc
SmallScaleVirions.doc
ssDNA.doc
sequencing.doc
micropan.doc
ELISA.doc
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List of documents (continued)
Contents
Preparation of large quantities of double-stranded circular
vector RF DNA for cloning. We no longer use the
CsCl/EtBr method even though it yields much purer RF
DNA
Special precautions for large-scale propagation of noninfective vectors fUSE1, fUSE3, fUSE5 and fUSE55
Cleavage of double-stranded vector at cloning sites and
removal of stuffer:
Construction of a random peptide library (RPL)
Construction of a natural peptide library (NPL)
Transfection of
Electrocompetent cells
naked DNA into
Electroporation
cells by
A homemade electroporation device
electroporation
designed with safety in mind
Large-scale, high-purity preparation of fd-tet-based virions
Large-scale, high-purity preparation of wild-type virions
Absorption spectrum and quantitation of filamentous phage
Titering infectious units of fd-tet-based phage as tetracycline
transducing units
Quantifying infectious units of wild-type phage as plaqueforming units (including blue plaques)
Quantitation of recombinant pVIII subunits from f88 vectors
by protein gel electrophoresis and western blotting
Extraction of double-stranded
from 200-ml cultures
replicative-form (RF) phage DNA from 7-ml cultures
fMCS1, a general utility, non-phage-display vector based on
fd-tet, with XhoI, BglII, PstI and PvuII cloning sites
Document
RFmaxiprep2.doc (QIAGEN
column); RFmaxiprep.doc
(CsCl/EtBr density gradient
centrifugation)
fUSE135propagation.doc
CutVector.DOC
RPLconstruct.DOC
NPLconstruct.doc
ElectrocompetentCells.doc
electroporation.doc
electroporator.doc
VirionPurification.DOC
WildtypeVirionPurification.doc
AbsorptionSpectrum.doc
TUtiter.doc
Plaques.doc
p8page.doc
RFmidiprep2.doc
RFminiprep2.doc
fMCS1.doc