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Cloning Procedures
Background
Procedures
Additional Sources

Introduction

Cloning methods (from The Protein Expression and Purification Facility of EMBL)

Getting started with molecular cloning (NEB)

Subcloning notebook (Promega)

Cloning and gene synthesis (Roche)

Using restriction enzymes

Plasmid Cloning by Restriction Enzyme Digest (AddGene)

Traditional cloning quick guide (NEB)

Cloning enzymes guide (Promega)

Optimizing restriction endonuclease reactions (NEB)

Restriction enzymes - technical tips (Roche)

Restriction enzyme troubleshooting guide (NEB)

Star activity (NEB)

Troubleshooting guide for cloning (NEB)

Heat inactivation (NEB)

Double digests (NEB)

Cleavage close to the end of DNA fragments (NEB)

DNA methylation & restriction enzymes

Alteration of apparent recognition specificities using methylases (NEB)

Effects of CpG methylation on restriction enzyme cleavage (NEB)

Dam and Dcm methylases in E-coli (NEB)

Making unmetylated DNA (NEB)

Modifying enzymes

DNA modifying enzymes

End-treatment tips (NEB)

PCR

Guidelines for PCR optimization with thermophilic DNA polymerases (NEB)

PCR review (Promega)

PCR troubleshooting guide (NEB)

Ligation

Tips for maximizing ligations efficiencies (NEB)

Troubleshooting guide for ligases (NEB)

Ligation independent cloning (EMBL)

 

Miscellaneous

Gel electrophoresis of DNA and RNA, Multiple DNA forms in gel,

Agarose gel resolution (NEB)

General molecular biology data (NEB)

Genetic markers (NEB)

Design parameters to control synthetic gene expression in E. coli (paper)

Let your fingers do the walking (an interactive tutorial)

Cloning Procedures

Preparing DNA
1. Miniprep tips (Hebrew)
2. Chloramphenicol - amplification of plasmids

PCR Cloning
1. Bio-X-act,
2. Platinum pfx,
3. Phusion

Non restriction cloning
1. Gibson assembly protocol

Ligation
DNA ligation tips (Hebrew)
Direct ligation with oligo

Recombination
1. Gateway

Mutagenesis:
1. Phusion
2. Quickchange
3. Quickchange multi

Miscellaneous Procedures

Modifying enzymes:
1. Klenow,
2. Mung Bean

PCR Screening
1. Colony PCR screening
2.GoTaq Green Master Mix

Additional Protocol Sites 
1.Molecular biology protocols from Protocol Online)
2. Cold Spring Harbor protocols


Commercial Sources

BioSupplyNet (searching for scientific supplies.)

NEB 

Fermentas  

Promega

Roche Applied Science

Takara

Sigma

Oligos Synthesis (Sigma/Genosis, Eisenberg Bros, MBC, Rhenium)

Vendors for DNA synthesis
When wishing to start a project, one of the main ways to obtain your gene of interest is to purchase it from one of the following vendors:

Protocol Sites

 

 



This site is maintained by Dr. Nurit Doron . Your comments are most welcome.

entries since October 2003

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