Boston University/Project Progress
From 2007.igem.org
(→Short-Term To-Do List) |
(→Short-Term To-Do List) |
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Need: | Need: | ||
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1)O/N of cells for Whole-Cell PCR (E.coli K12 for lacZ or Shewie for GTFs) | 1)O/N of cells for Whole-Cell PCR (E.coli K12 for lacZ or Shewie for GTFs) | ||
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2)O/N of cells for competency then transformation (E.coli to get plasmid with just lacZ) | 2)O/N of cells for competency then transformation (E.coli to get plasmid with just lacZ) | ||
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3)Selection plates (gent for plasmid pJQ200) | 3)Selection plates (gent for plasmid pJQ200) | ||
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Whole-Cell PCR (3 hours) | Whole-Cell PCR (3 hours) | ||
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-prepare agarose gel | -prepare agarose gel | ||
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Gel electrophoresis with SYBR safe (1 hour) | Gel electrophoresis with SYBR safe (1 hour) | ||
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-prepare Qiagen PCR Cleanup Kit | -prepare Qiagen PCR Cleanup Kit | ||
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Qiagen PCR Cleanup (0.5 hour) | Qiagen PCR Cleanup (0.5 hour) | ||
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-prepare digestion plasmid | -prepare digestion plasmid | ||
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Prepare digestion of PCR products (0.5 hour) | Prepare digestion of PCR products (0.5 hour) | ||
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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) | ||
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-prepare ligation | -prepare ligation | ||
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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) | ||
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-warm plates | -warm plates | ||
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Plate cells | Plate cells | ||
Revision as of 16:53, 16 July 2007
Contents |
What We've Accomplished
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
pTrcHis TOPO TA Expression Kit Cloning Protocol
Using NEBCutter for checking specific restriction enzymes against a sequence
Procedure for 50 mL E. Coli mixture in Zymo Broth or SOB
