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Team:TU-Delft/Protocol 7
From 2013.igem.org
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Revision as of 13:34, 1 October 2013
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
Gel Extraction Procedure
Requirements:
- Scalpel
- Pipettes
- Microcentrifuge tubes
- QIA quick column and collection tubes
- Buffer PE
- Buffer QG
- Sterile MilliQ
Prodecure:
- Excise the DNA fragment from the agarose gel with a clean scalpel.
- Weigh the gel slice in a tube. Add 3 volumes of Buffer QG to 1 volume of the gel.
- Incubate at 50 ⁰C for 10 mins (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2-3 mins during incubation.
- After the gel slice has dissolved completely check that the colour of the mixture is yellow.
- Place a QIA quick spin column in 2mL collection tube and centrifuge for 1 min.
- Discard the flow through and place QIA quick column back in same collection tube.
- To wash, add 0.75 mL of Buffer PE to the QIA quick column and centrifuge for 1 min at 13000 rpm.
- Discard the flow through and centrifuge for an additional 1 min at 13000 rpm to remove the remaining ethanol.
- Place the column on a clean microcentrifuge tube.
- Add 40-50 µL of milliQ sterile H2O.
- Incubate in oven for 2 mins and then centrifuge again for 1 min.
- Measure on Nanodrop for the concentration of the DNA.
- Gel Extraction Kit ProtocolQIAquick Spin Handbook, Version 3, 2001.[Online]. Available From: http://devbio.wustl.edu/krolllab/Kroll_Lab_Protocols/Molecular%20Biology%20protocols/Cloning%20protocols%20folder/Gel%20extraction-Qiagen.pdf
