IGEM:IMPERIAL/2007/Notebook/General Protocols

Preparation of Media
Summary: preparing nutrient broths where bacteria grow

Transformation
Summary: inserting plasmids into bacterial cells

Induction of T7 polymerase with IPTG
Summary: inducing the production of T7 polymerase in BL21 cells with IPTG

Plasmid Preparation
Summary: extracting plasmid DNA from bacterial cells

Solutions Required
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Tris-Acetate
To make 1 L of 50x Tris-acetate solution:
 * 242 g Tris base
 * 57.1 ml glacial acetic acid
 * 1) Add H20 to 1 L

Glucose/Tris/EDTA Solution

 * 50 mM glucose
 * 25 mM Tris·Cl, pH 8.0
 * 10 mM EDTA
 * 1) Autoclave and store at 4°C

NaOH/SDS Solution
[Note: Prepare NaOH/SDS solution immediately before use]
 * 0.2 M NaOH
 * 1% (wt/vol) sodium dodecyl sulfate (SDS)

Potassium Acetate Solution
5 M potassium acetate solution, pH 4.8 }}
 * 29.5 ml glacial acetic acid
 * 1) Add KOH pellets until solution reaches pH 4.8
 * 2) Add H2O to 100 ml
 * 3) Store at room temperature (do not autoclave)

Restriction Digest
Summary: using enzymes to cut DNA on specific sites - this is used to cut at the suffix or prefix of the biobrick plasmids

Solutions Required
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Tris/EDTA Buffer

 * 10 mM Tris×Cl
 * 1 mM EDTA, pH 8.0

Tris×Cl
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 * Tris×Cl [tris(hydroxymethyl)aminomethane], 1 M
 * 1) Dissolve 121 g Tris base in 800 ml H2O
 * 2) Adjust to desired pH with concentrated HCl
 * 3) Mix and add H2O to 1 liter

Ligation
Summary: sticking the DNA together

Agarose Electrophoresis
Summary: using an electric field to seperate DNA of different sizes in a gel

Solutions Required
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Tris-Borate-EDTA
To make 10x TBE: }}
 * 108g Tris
 * 55g boric acid
 * 7.44g di-sodium EDTA
 * Make up to 1 litre with ddH2O

DNA Extraction/Purification
Summary: extracting and purifying the DNA from the gel or reaction mixture