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)

(573) 882-0123 (fax)

smithgp@ missouri.edu (e-mail)

 

Update History

 

All but 10 of the documents in this webpage (see the table of descriptions below) were updated or posted anew in the period 2/28/06 to 3/4/06; a list of the most recent document modification dates can be found in ModificationDates.doc. As newly modified documents are posted subsequent to that time, they will be listed below, the most recent postings at the top.

Posted 3/22/06 5:00 PM Central Standard Time: Minor corrections to stdpreps.doc, vectors.doc; see below under Distribution Policy for newly posted Kit5.doc.

Posted 3/9/06 Noon Central Standard Time: Changes to AffinitySelection.doc, vectors.doc, stdpreps.doc and KitRequest.doc describing use of (and allowing requests for) fd-cat internal enrichment control phage

Posted 3/5/06 2:00 AM Central Standard Time: RPLconstruct.DOC updated (minor change: graph inserted at last step)

 

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 materials will have labels that reference a distribution document called Kit#.doc, where # is a serial number; that document will contain our internal references for the distributed materials, and may be useful information to us in answering questions. Here are links to the distribution documents and their posting dates.

Posted 3/22/06: Kit5.DOC

 

A basic review of phage display from Chemical Reviews 1997: PetrenkoSmithChemReviews.PDF

 

Standard Preparations and Procedures are referred to in italics

 

Throughout the documents on this webpage, 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 Webpage Contains Links to Documents That Describe Our Phage-Display System and How to Use It

 

This document contains numerous links to Microsoft Word documents (as well as to the pdf document above), each of which details some specific aspect of the phage-display system.  In order to be accessible, a document must previously have been downloaded from this website, using the links on this page.  These documents cross-reference each other extensively through local hyperlinks (using relative paths), which will work only if all the documents are downloaded to 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, you should certainly download MainIndex.doc (basically a Microsoft Word version of the present webpage, containing brief descriptions of and hyperlinks to all the other Word documents) and the above-mentioned stdpreps.doc (containing standard preparations and procedures referenced in almost all the other documents).

 

Contents

Document

An index containing descriptions of and hyperlinks to the other documents on this web site; bascially a Microsoft Word version of the current webpage

MainIndex.doc

Standard preparations (solutions, media, etc.) and procedures referenced in almost all the other documents

stdpreps.doc

Description and confirmation of E. coli strains

Strains.doc

Description of fUSE vectors (display on gene III protein) and f88 vectors (display on gene VIII protein)

vectors.doc

Random peptide libraries in fUSE5 and f88-4 vectors

Descriptions of libraries

library.doc

Sequences of insert regions

libeseq.doc

Form for requesting vectors, libraries or strains

KitRequest.doc

Amplifying a large phage-display library without losing diversity

AmplifyingLibrary.doc

Biotinylation of a receptor for use in affinity selection and binding assays

biotinylation.doc

Affinity selection (“biopanning”): using a target selector (receptor, antibody, other binding molecule) to select binding clones from large libraries

AffinitySelection.doc

1-ml scale propagation and processing of clones for sequencing and binding assays (we never do this any more)

Propagation_1ml.doc

7-ml scale propagation and partial purification of virions SmallScaleVirions.doc
Extraction of ssDNA from virions for sequencing or other purposes ssDNA.doc

Manual procedure for sequencing inserts in large numbers of clones (we never do this any more)

sequencing.doc

Binding assays for confirming affinty of phage clones for target receptor

Phage capture assay (miniaturized analytical version of affinity selection, in which the input is individual clones rather than complex populations)

micropan.doc

Quantifying binding of selector to phage clones immobilized on the surface of plastic wells

ELISA.doc

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

RFmaxiprep2.doc (QIAGEN column); RFmaxiprep.doc (CsCl/EtBr density gradient centrifugation)

Special precautions for large-scale propagation of non-infective vectors fUSE1, fUSE3, fUSE5 and fUSE55

fUSE135propagation.doc

Cleavage of double-stranded vector at cloning sites and removal of stuffer

CutVector.DOC

Construction of a random peptide library (RPL)

RPLconstruct.DOC

Construction of a natural peptide library (NPL)

NPLconstruct.doc

Transfection of naked DNA into cells by electroporation

Electrocompetent cells

ElectrocompetentCells.doc

Electroporation

electroporation.doc

A homemade electroporation device designed with safety in mind

electroporator.doc

Large-scale, high-purity preparation of fd-tet-based virions

VirionPurification.doc

Large-scale, high-purity preparation of wild-type virions

WildtypeVirionPurification.doc

Absorption spectrum and quantitation of filamentous phage

AbsorptionSpectrum.doc

Titering infectious units of fd-tet-based phage as tetracycline transducing units

TUtiter.doc

Quantifying infectious units of wild-type phage as plaque-forming units (including blue plaques)

Plaques.doc

Quantitation of recombinant pVIII subunits from f88 vectors by protein gel electrophoresis and western blotting

p8page.doc

Extraction of double-stranded replicative-form (RF) phage DNA

from 200-ml cultures

RFmidiprep2.doc

from 7-ml cultures

RFminiprep2.doc

fMCS1, a general utility, non-phage-display vector based on fd-tet, with XhoI, BglII, PstI and PvuII cloning sites

fMCS1.doc