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Team:TU-Delft/Protocol 3
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Revision as of 14:57, 3 October 2013 by MaithiliKrishnan (Talk | contribs)
Protocols
Our project deals with E.coli cells which sense Auto-inducing peptides (AIPs) from the Staphylococcus aureus and starts producing Antimicrobial peptides in order to kill the Staphylococcus aureus. Different protocols used during the project are described below.
- Transforming Parts from Distribution kit
- Growing the Single Colonies from the Agar Plates
- Making glycerol stocks
- Plasmid Purification Protocol
- Restriction digestion
- Ligation
- Gel Extraction Procedure
- PCR Purification
- Tricine Gels
- General Peptide Production
- SUMO cleavage
- Lysis Protocol
- AIP Sensing Protocol
- Gene Design
- Primer Design
Making glycerol stocks
Requirements:
- bacterial culture
- LB medium
- 80% glycerol
- centrifuge
Prodecure:
- Take 5 mL bacterial cells from the Erlenmeyer of a freshly grown culture and spin in a 15 mL tube for 10 minutes at 2,000 rpm (Eppendorf centrifuge).
- Decant the supernatant without disturbing the pellet.
- Pipet on the pellet 0.5 mL 1% LB medium and 0.5 mL 80% glycerol and mix by vortexing and save in -80 °C freezer.
The glycerol stock can be used whenever required, by just adding 0.5 mL of stock into 5 mL of freshly prepared media.
