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Team:Goettingen/protocol protein
From 2014.igem.org
Overview
PCR Methods
Plasmid Construction
- Restriction of DNA
- Ligation of DNA fragments
- BP recombination reaction
- LR recombination reaction
- SEAMLESS Cloning
- Peptide Library construction
Plasmid Transformation
- E.coli competent cells
- Plasmid isolation (E.coli)
- E.coil transformation
- Plasmid isolation (Yeast)
- Yeast transformation
Colony Scanning
Protein Assessment
In vivo tests
SDS-PAGE
1. Clean the glass plates, combs and mats with EtOH and assemble the glass plates.
2. Wear gloves while making the gels (Acrylamid is neurotoxic). Mix the ingredients of the running gel and pour the mixture between the assembled glass plates. Overlay the running gel with Ethanol or Isopropanol to make sure that surface is horizontal.
3. After polymerisation, remove the alcohol and dry with Whatman-paper. Mix the ingredients and pour it on top of the running gel. Insert a comb immediately.
4. After polymerisation, clamp the gel into the electrophoresis chamber and fill the chamber with 1x running buffer. Remove the comb and wash the wells with running buffer using a pipette.
5. The protein samples (up to 15 µl) are mixed with SDS-loading dye (2.5 µl) and incubated at 95°C for 10 minutes.
6. Briefly centrifuge the samples.
7. Load the samples with a pipette into the wells of the gel. Do not forget the MARKER.
8. Run the gel at 80 V for 10 minutes (sample will pass the stacking gel) and then turn up to 120 V.
9. Pry the plates apart with a gel spacer. Gels can be stained with Coomassie (or Silver Stain) to visualize the proteins.
