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Revision as of 16:53, 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 33:		Line 33:
	Whole-Cell PCR (3 hours)		Whole-Cell PCR (3 hours)
-	-prepare agarose gel	+	::prepare agarose gel
	Gel electrophoresis with SYBR safe (1 hour)		Gel electrophoresis with SYBR safe (1 hour)
-	-prepare Qiagen PCR Cleanup Kit	+	::prepare Qiagen PCR Cleanup Kit
	Qiagen PCR Cleanup (0.5 hour)		Qiagen PCR Cleanup (0.5 hour)
-	-prepare digestion plasmid	+	::prepare digestion plasmid
	Prepare digestion of PCR products (0.5 hour)		Prepare digestion of PCR products (0.5 hour)
Line 47:		Line 47:
	Digestion of PCR products and plasmid (3 hours)		Digestion of PCR products and plasmid (3 hours)
-	-prepare Qiagen PCR Cleanup Kit	+	::prepare Qiagen PCR Cleanup Kit
-	-prepare agarose gel (SYBR safe)	+	::prepare agarose gel (SYBR safe)
	Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)		Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour)
-	-prepare Qiagen Gel Extraction Kit	+	::prepare Qiagen Gel Extraction Kit
	Qiagen Gel Extraction of plasmid from gel (0.5 hour)		Qiagen Gel Extraction of plasmid from gel (0.5 hour)
-	-prepare ligation	+	::prepare ligation
	Ligation (1 hour)		Ligation (1 hour)
-	-prepare competent cells from O/N culture	+	::prepare competent cells from O/N culture
	Transformation (0.5 hour)		Transformation (0.5 hour)
-		+	::warm plates
-	-warm plates	+
	Plate cells		Plate cells

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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

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