From 2007.igem.org

Contents 1 What We've Accomplished 2 Materials We Need 3 Short-Term To-Do List 4 Protocols 5 Question and Answer

What We've Accomplished

Plasmid Choice

Restriction Enzyme Choice

Primer Design

Making Shewanella Competent and Transforming Plasmid

Materials We Need

Error-Prone PCR: From CAB(?)

Ligases: Need to Buy

Short-Term To-Do List

EDIT THE WIIIIKIIII

Clone lacZ promoter into pJQ200:

Tuesday 7/17/07 (11 hour protocol)

Need:

1)O/N of cells for Whole-Cell PCR (E.coli K12 for lacZ or Shewie for GTFs)

2)O/N of cells for competency then transformation (E.coli to get plasmid with just lacZ)

3)Selection plates (gent for plasmid pJQ200)

Whole-Cell PCR (3 hours)

-prepare agarose gel

Gel electrophoresis with SYBR safe (1 hour)

-prepare Qiagen PCR Cleanup Kit

Qiagen PCR Cleanup (0.5 hour)

-prepare digestion plasmid

Prepare digestion of PCR products (0.5 hour)

Digestion of PCR products and plasmid (3 hours)

-prepare Qiagen PCR Cleanup Kit

-prepare agarose gel (SYBR safe)

Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)

-prepare Qiagen Gel Extraction Kit

Qiagen Gel Extraction of plasmid from gel (0.5 hour)

-prepare ligation

Ligation (1 hour)

-prepare competent cells from O/N culture

Transformation (0.5 hour)

-warm plates

Plate cells

Protocols

Calcium Chloride/Heat Shock Plasmid Transformations Protocol

Filter Cojugation Protocol

pTrcHis TOPO TA Expression Kit Cloning Protocol

Using NEBCutter for checking specific restriction enzymes against a sequence

Making an Electrophoresis Gel

Procedure for 50 mL E. Coli mixture in Zymo Broth or SOB

Question and Answer

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