Boston University/Project Progress



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


Clone lacZ promoter into pJQ200:

Tuesday 7/17/07 (11 hour protocol)


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


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