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)
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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
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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
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