Molecular biology

From CSBLwiki

Contents 1 Reagents & Protocols 1.1 Recipes 1.2 Protocols 1.2.1 LIC cloning 1.2.2 Conjugation 1.2.3 Gene replacement 2 Tools 2.1 webtools 2.2 softwares 3 Links

Reagents & Protocols

Recipes

• Chemicals
• Commonly used reagents and equipment in molecular biology (PDF)
• Concentration of antibiotics commonly used
• For DNA extraction (to remove some protein contaminants & inactivate enzymes): Phenol:Chloroform=1:1(v:v) solution
• DNA sequencing check list

1. quality score
2. good sequencing result vs. bad sequencing result (see below example)

good vs. bad sequencing example

Protocols

• PCR - General rules for primer design
• Genomic DNA extraction
• Competent cell preparation BSGC protocol (PDF)
  • Easy & rapid competent cell preparation reference link - by HJKo
• Electrocompetent cell Protocol (PDF)

LIC cloning

• Reference:

1. Error fetching PMID 2235490: [LIC]

• LIC cloning introduction LIC introduction - Ligation Independent Cloning
  • LIC cloning protocol (Berkeley Structural Genomics Center)
    • Vector preparation
    • Insert preparation
    • LIC transformation:
      • mix insert and vector in appropriate amounts
      • incubate 5~30 min(20 min) at room temperature
      • do transformation in E. coli (recommend to use electro-competent cells)

Conjugation

• Donor: E. coli S17-1

Gene replacement

• E. coli knockout by the Wanner method (Lambda Red Recombinase)

Tools

webtools

1. Oligo Tm calculation

softwares

1. NIH Image: Gel analysis software
2. Serial Cloner: DNA sequence management (PCR, restriction maps, etc.)
3. BioX (Mac OS X only)
4. Biological sequence editor Bioedit
5. Jmol

Links

• NCBE practical protocols: very good illustration for undergraduate students