Team:Thessaloniki/Experiments

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• Solutions Preparation
• Bacterial Cultures
• DNA Isolation
• DNA Amplicifation
• Electrophoresis
• Cloning
• Cell-Free Protein Expression
• EXPAR

Experiments

During our Wet Lab, we used a series of protocols for our experiments that helped us shape our project. In hope that these protocols might help future iGEM teams we attach them below!

Preparing Solutions

• Preparation of TE Buffer

• Tris-HCI (1M) recipe

• Preparation of Glycerol 50%

Bacterial Cultures

• Preparing LB-agar

• Preparing LB-agar plates

• Preparation of Chemically Competent Cells

• Preparation of glycerol stock

• Inoculation of liquid bacterial cultures

• Streaking and Isolating Bacteria on an LB Agar Plate

DNA Isolation

• Plasmid DNA Isolation with miniprep

• Gel Extraction

• PCR Cleanup

DNA Amplification

• Primer Preparation

• PCR using Q5 High Fidelity

Electrophoresis

• Preparation of 10x TAE buffer

• Electrophoresis

Cloning

• Digestion of g-Blocks

• Digestion of pSB1C3 Vector

• Dephosphorylation of linearized vector

• Ligation with T4 ligase

• Transformation of competent cells

• Diagnostic Restriction Digestions

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• PCR cloning

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Cell-Free Protein Expression

• Resuspension of gBlocks

• In vitro Expression with Cell Free System

EXPAR

• Primer Ligation with T4

• EXPAR DNA Protocol 37 ℃

• EXPAR DNA Protocol 65 ℃

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• EXPAR RNA Protocol 37 ℃

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