Difference between revisions of "Davidson Missouri W/Davidson Protocols"

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* What is the list of DNA parts that we will need for the first stage, second stage, entire project?<br>
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'''A. General Lab Information'''
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*<u>ERIN and SABRIYA</u> Which DNA parts exist in the registry?[[Answer1]][[More Parts]]<br>
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# [http://www.bio.davidson.edu/courses/molbio/labnotebook.html How to Keep a Lab Notebook]
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* <u>ERIN and SABRIYA</u>Which DNA parts will have to be designed?  Will they be synthesized or produced by PCR? <br>
 
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[[loxP sites]] [[Hin/Hix]] [[CRE gene]] [[Degradation Tags]]
 
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/reagents.html Common molecular reagents]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/reagents.html Common molecular reagents]
 
# [http://parts.mit.edu/registry/index.php/Assembly:Standard_assembly Standard Assembly]
 
# [http://parts.mit.edu/registry/index.php/Assembly:Standard_assembly Standard Assembly]
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#[http://www.bio.davidson.edu/courses/Molbio/Protocols/ORIs.html '''Compatibility of Plasmids''']
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# [http://partsregistry.org/Help:BioBrick_Prefix_and_Suffix BioBrick Ends]
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/anneal_oligos.html Building dsDNA with Oligos]
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/ORIs.html '''Compatibility of Plasmids''']
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/pcr.html Setting up PCR mixtures]
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/clean_short.html Ethanol Precipitate DNA (short protocol)]
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/magnesium.html PCR and Mg<sup>2+</sup> concentration]
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# [[glycerolstocks How to Make Glycerol Stocks of Bacteria]]
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#[http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html Clean and Concentrate DNA (after PCR, before digestion)]
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'''B. Gel Electrophoresis and Purification'''
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/pourgel.html Pouring an agarose gel]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/pourgel.html Pouring an agarose gel]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/molwt.html Calculate MWs]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/molwt.html Calculate MWs]
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/digestion.html Digest DNA with restriction enzymes]
 
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# [[Double Digest Guide]]
 
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/clean_short.html Ethanol Precipitate DNA (short protocol)]
 
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/gels2002/1kbladder.pdf 1kb MW markers]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/gels2002/1kbladder.pdf 1kb MW markers]
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/SAP.html Shrimp Alkaline Phosphatase]
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/MN_gelpure.html Macherey-Nagel Gel Purification (improved 260/230 ratios)l]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Qiagen_gelpure.html Qiagen QIAquick Gel Purification]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Qiagen_gelpure.html Qiagen QIAquick Gel Purification]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/QIAQuick_recycle.html Qiagen QIAquick Column Regeneration Protocol]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/QIAQuick_recycle.html Qiagen QIAquick Column Regeneration Protocol]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/gelpure.html ElectroElute Gel Purification]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/gelpure.html ElectroElute Gel Purification]
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'''C. Digestion, Ligation, Transformation'''
 +
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/digestion.html Digest DNA with restriction enzymes]
 +
# [[Davidson Missouri W/Double Digest Guide| Double Digest Guide]]
 +
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/SAP.html Shrimp Alkaline Phosphatase]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/ligation.html Ligation Protocol]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/ligation.html Ligation Protocol]
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Tranformation_list.html Choices for Transformation: Heat Shock vs. Zyppy]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Promegacompcells.pdf Heat Shock Transformation] OR [http://www.bio.davidson.edu/courses/Molbio/Protocols/transformation.html Short version of Heat Shock]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Promegacompcells.pdf Heat Shock Transformation] OR [http://www.bio.davidson.edu/courses/Molbio/Protocols/transformation.html Short version of Heat Shock]
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#[http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Transformation]
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Transformation]
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#[http://www.bio.davidson.edu/courses/Molbio/Protocols/ColonyPCR_Screening.html Colony PCR to Screen for Successful Ligations]
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# [[TSS Competent Cells|TSS Competent Cell Transformation]]
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 +
'''D. Minipreps'''
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/MiniPrep_list.html Choices for Mini-Preps: Promega vs. Zyppy]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/miniprepPrmega.html Promega miniprep]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/miniprepPrmega.html Promega miniprep]
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#[http://www.bio.davidson.edu/courses/Molbio/Protocols/Tranformation_list.html Choices for Transformation: Heat Shock vs. Zyppy]
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html Zippy Miniprep]
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#[http://www.bio.davidson.edu/courses/Molbio/Protocols/MiniPrep_list.html Choices for Mini-Preps: Promega vs. Zyppy]
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 +
'''E. Making New Parts and PCR'''
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/anneal_oligos.html Building dsDNA with Oligos]
 +
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/pcr.html Setting up PCR mixtures]
 +
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/magnesium.html PCR and Mg<sup>2+</sup> concentration]
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# [[Davidson Missouri W/Primer_dimer| Making dsDNA Using Primer Dimers]]
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# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html Clean and Concentrate DNA (after PCR, before digestion)]
 +
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/ColonyPCR_Screening.html Colony PCR to Screen for Successful Ligations]
 +
 
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'''F. Expression of Phenotypes'''
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# [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC106306/pdf/am002240.pdf Using degradation tags on proteins such as GFP]
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# [[Genomic Insertion Protocol|Genomic Insertion Protocol]]
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# When inducing with IPTG, use '''3 µL of stock''' (0.2 g/mL = 20% w/v) '''to every 1 mL''' of LB or other liquid.
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# When inducing with Arabinose, use "2 µL of stock" (10% w/v L-Arabinose) "to every 1 mL" of LB or other liquid.
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# When inducing with 3OC6 (HSL), use a '''2000 fold dilution of a 10 mg/mL stock solution'''. We have dissolved in EtOH which is not the best - degrades with time. Keep this cold.
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# [http://partsregistry.org/AHL List of auto-inducers and their catalog numbers]
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 +
 
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'''G. Computer Tools We Use'''
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# [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Optimize your Gel]
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#[http://genedesign.thruhere.net/gd/ Gene Design (Boeke Lab at JHU)]
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# [http://gcat.davidson.edu/iGEM07/genesplitter.html Gene Splitting Web Site]
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# [http://gcat.davidson.edu/iGEM08/bbprimer.html PCR Primers w/ BioBricks]
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# [http://www.promega.com/biomath/calc11.htm Promega T<sub>m</sub> Calculator]
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# [http://gcat.davidson.edu/iGem10/index.html Oligator making dsDNA from oligos] by Steph and Stephen
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# [http://gcat.davidson.edu/IGEM06/oligo.html Lance-olator Oligos for dsDNA assembly]
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# [http://gcat.davidson.edu/GCATalog Access the GCAT-alog of Davidson and MWSU DNA Freezer Stocks]
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# [[Sequencing at MWG Operon| Sequencing at MWG Operon]]
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# [[Sequencing at Agencourt| Sequencing at Agencourt Bioscience]]
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# [[Davidson Missouri W/CUGI_Seuqencing| Sequencing at CUGI]]
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# [http://gcat.davidson.edu/GcatWiki/images/3/3b/Ape_protocol.pdf Analyzing Sequences with aPe]
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# [[Using Apes (A Plasmid Editor)]]
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# [http://72.22.219.205/sequence VeriPart for DNA sequences of Registry Parts]
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# [http://gcat.davidson.edu/igem10/opt/opt_index.html The Optimus for optimizing codons]

Latest revision as of 01:39, 3 August 2010

A. General Lab Information

  1. How to Keep a Lab Notebook
  2. Common molecular reagents
  3. Standard Assembly
  4. BioBrick Ends
  5. Compatibility of Plasmids
  6. Ethanol Precipitate DNA (short protocol)
  7. glycerolstocks How to Make Glycerol Stocks of Bacteria

B. Gel Electrophoresis and Purification

  1. Pouring an agarose gel
  2. Calculate MWs
  3. 1kb MW markers
  4. Macherey-Nagel Gel Purification (improved 260/230 ratios)l
  5. Qiagen QIAquick Gel Purification
  6. Qiagen QIAquick Column Regeneration Protocol
  7. ElectroElute Gel Purification

C. Digestion, Ligation, Transformation

  1. Digest DNA with restriction enzymes
  2. Double Digest Guide
  3. Shrimp Alkaline Phosphatase
  4. Ligation Protocol
  5. Choices for Transformation: Heat Shock vs. Zyppy
  6. Heat Shock Transformation OR Short version of Heat Shock
  7. Zippy Transformation
  8. TSS Competent Cell Transformation

D. Minipreps

  1. Choices for Mini-Preps: Promega vs. Zyppy
  2. Promega miniprep
  3. Zippy Miniprep

E. Making New Parts and PCR

  1. Building dsDNA with Oligos
  2. Setting up PCR mixtures
  3. PCR and Mg2+ concentration
  4. Making dsDNA Using Primer Dimers
  5. Clean and Concentrate DNA (after PCR, before digestion)
  6. Colony PCR to Screen for Successful Ligations

F. Expression of Phenotypes

  1. Using degradation tags on proteins such as GFP
  2. Genomic Insertion Protocol
  3. When inducing with IPTG, use 3 µL of stock (0.2 g/mL = 20% w/v) to every 1 mL of LB or other liquid.
  4. When inducing with Arabinose, use "2 µL of stock" (10% w/v L-Arabinose) "to every 1 mL" of LB or other liquid.
  5. When inducing with 3OC6 (HSL), use a 2000 fold dilution of a 10 mg/mL stock solution. We have dissolved in EtOH which is not the best - degrades with time. Keep this cold.
  6. List of auto-inducers and their catalog numbers


G. Computer Tools We Use

  1. Optimize your Gel
  2. Gene Design (Boeke Lab at JHU)
  3. Gene Splitting Web Site
  4. PCR Primers w/ BioBricks
  5. Promega Tm Calculator
  6. Oligator making dsDNA from oligos by Steph and Stephen
  7. Lance-olator Oligos for dsDNA assembly
  8. Access the GCAT-alog of Davidson and MWSU DNA Freezer Stocks
  9. Sequencing at MWG Operon
  10. Sequencing at Agencourt Bioscience
  11. Sequencing at CUGI
  12. Analyzing Sequences with aPe
  13. Using Apes (A Plasmid Editor)
  14. VeriPart for DNA sequences of Registry Parts
  15. The Optimus for optimizing codons
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