Notebook

From early June to late October we have worked hard in the lab to realize Esther. Here, you can find all of the protocols used, a timeline of important points in the process, as well as the lab notes themselves.

Dictionary

During our laboratory work, we assigned nicknames to our BioBricks for the sake of simplicity. This table will allow the reader to understand what BioBrick we are mentioning in our notebook.

Notebook nameWiki nameRegistry Link
BB1C-protein Characterization Construct (Lysogenic module)http://parts.igem.org/Part:BBa_K3024004
BB1.2Inducible C-protein construct - Regulate lysogenic modulehttp://parts.igem.org/Part:BBa_K3024009
BB2Inducible cox characterization construct (Lytic module)http://parts.igem.org/Part:BBa_K3024011
BB3Phage genome simulation modulehttp://parts.igem.org/Part:BBa_K3024012

Protocols

PCR

Taq Colony PCR Protocol.pdf

Q5 PCR Protocol.pdf

Digestion

Digestion Protocol AraC3 Vector and BioBricks.pdf

Digestion Protocol C3 Vector and BioBricks.pdf

Ligation and Transformation

Ligation and Transformation Protocol AraC3 and C3 vectors.pdf

HIFI Cloning

Manufacturers protocol:

NEBuilder HiFi DNA Assembly Reaction Protocol

Alterations on the manufacturers protocol:

Create inserts with complementary ends to the vector, by either amplifying the vector or the inserts. a Total amount of max 200fmol DNA, with 50ng of Vector, is mixed with the HIFI master mix and incubated at 50°C for 15 minutes. Of the 20µl ligation reaction, 2µl were mixed with 6µl of water and 2µl 5x KCM. After one minute on Ice, this mix was used for transformation by heat-shock with 10µl of KCM competent cells.

HIFI cloning.pdf

Fluorescence spectrometry

Fluorescence Spectrometry Method.pdf

Flow cytometry

Flow Cytometry Protocol.pdf

Absorbance spectrometry

Absorbance Spectrometry.pdf

KCM Competent Cells

KCM Competent Cells Protocol.pdf

Western blot

Western blot Protocol.pdf

Overlap Extension

OE-PCR protocol.pdf

Overlap Extension Protocol.pdf

Q5 Site Directed Mutagenesis

Q5® Site-Directed Mutagenesis Kit Protocol (E0554)

Workflow

June

July

August

September

October

Detailed lab notes

Notebook Plasmid A and Plasmid B Characterization (Benchling).pdf

HiFI cloning (Benchling).pdf