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DNA Protocols

This section contains just about everything you need to know to assemble plasmids for the expression of cloned genes encoding proteins, fusion proteins and mutant proteins using cell free systems. Some of the protocols are pretty similar to those in standard cloning manuals. However, even for these we have attempted to illustrate protocols in a form that is more convenient.


DNA ISOLATION AND PLASMID PURIFICATION

Phenol extraction and ethanol precipitation.

Large Scale Plasmid Prep for 100 ML Superbroth Cultures

Plasmid Miniscreen Protocol (Boil Prep)

Alkaline Lysis Miniprep

Alkaline Lysis Miniprep Modified 1

Alkaline Lysis Miniprep Modified 2

Amplification of plasmids with chloramphenicol

Single-Strand DNA Prep. Large Scale

Titre of Bacteriophage

Large Scale Bacteriophage Prep


LIGATION


RESTRICTION DIGESTS

Restriction Endonuclease Digestion

Large Scale Digest Prep for Construction

AGAROSE GEL ELECTROPHORESIS

ISOLATION OF DNA FRAGMENTS

Elution of DNA from Agarose Gels - Glassmilk Method

Elution of Small Fragments of DNA from Agarose Gels(<500bp)

Gel Purification of DNA from Agarose Gels by Centrifugation


POLYMERASE CHAIN REACTION (PCR)

Polymerase Chain Reaction

Melting Temperature Calculation


DNA MODIFYING ENZYMES

S1 Nuclease Digest of ssDNA

Alkaline Phosphatase Digestion of DNA

BAP

CIP


MANUAL SEQUENCING METHODS (ANCIENT HISTORY)

NEB Sequencing (Vent Polymerase)-- Preparing Samples to be Sequeneced

NEB Extension/Termination 35S-dATP Sequencing

Sequencing Gels

Sequencing Reaction: (Sequenase)


QUANTIFICATION

Fluorometer Procedures


USING OLIGONUCLEOTIDES

Melting Temperature Oligos

Phosphorylation with PNK


END REPAIR OF DNA FRAGMENTS

T4 Polymerase for Blunting 3' Extensions

Repair of Sticky Ends with Klenow (Large) Fragment of DNA Polymerase I

Mung Bean Nuclease Digestion



Please feel free to try any of our protocols.  They are available in web and Adobe Acrobat format.  All that we ask that you credit us (or the original author) when using them.  This document is in a permanent state of flux - no protocol is ever really perfect and therefore it is never finished!