From 2007.igem.org

Revision as of 16:51, 16 July 2007 (view source) Linger (Talk | contribs) (→Short-Term To-Do List) ← Older edit		Revision as of 16:53, 16 July 2007 (view source) Linger (Talk | contribs) (→Short-Term To-Do List) Newer edit →
Line 17:		Line 17:
	== Short-Term To-Do List ==		== Short-Term To-Do List ==
	EDIT THE WIIIIKIIII		EDIT THE WIIIIKIIII
-	Clone lacZ promoter into pJQ200	+
		+	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 ==		== Protocols ==

---

Revision as of 16:53, 16 July 2007

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

Back