Davidson Protocols

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A. General Lab Information

  1. How to Keep a Lab Notebook
  2. Common molecular reagents
  3. Open Access Libraries
  4. Standard Assembly
  5. BioBrick Ends
  6. Compatibility of Plasmids
  7. Ethanol Precipitate to clean DNA (short protocol)
  8. glycerolstocks How to Make Glycerol Stocks of Bacteria
  9. Pipet Tip Olympic Records

B. Gel Electrophoresis and Purification

  1. Pouring an agarose gel
  2. Calculate MWs
  3. 1kb Plus 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
  9. Golden Gate Assembly protocol
  10. Electroporation_Transformation developed at MWSU

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. Annealing_Oligos_for_Cloning Calculate how to mix boiled oligos with 50 ng of receiving plasmid.
  3. The Loligator
  4. Setting up PCR mixtures
  5. PCR and Mg2+ concentration
  6. isolate genomic DNA from a single hair follicle
  7. Prepare PCR product for sequencing after clean and concentration procedure (for Bio113 lab use)
  8. Making dsDNA Using Primer Dimers
  9. Clean and Concentrate DNA with spin column (after PCR, before digestion)
  10. Colony PCR to Screen for Successful Ligations
  11. Golden Gate Assembly protocol
  12. How many clones should I screen?
  13. TAS2R38 PCR amplification

F. Expression of Phenotypes

  1. Using degradation tags on proteins such as GFP
  2. Genomic Insertion Protocol
  3. M9CA media (J864-100G from Amresco) + 2mM MgSO4 (2mL/L of 1 M MgSO4) + 0.1 mM CaCl2 (0.1 mL/L 1 M CaCl2) and optional 0.2% glucose (10 mL/L 20% stock solution).
  4. 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.
  5. When inducing with Arabinose, use "2 µL of stock" (10% w/v L-Arabinose) "to every 1 mL" of LB or other liquid.
  6. 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.
  7. List of auto-inducers and their catalog numbers
  8. Synergy Machine Protocol

G. Golden Gate Shuffling

  1. Media:DNAShuffling.docx

H. 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. NEB Phusion Tm Calculator
  7. Oligator making dsDNA from oligos
  8. The Loligator
  9. How many clones should I screen?
  10. Lance-olator Oligos for dsDNA assembly old version, we recommend Oligator now
  11. Access the GCAT-alog of Davidson and MWSU DNA Freezer Stocks
  12. Sequencing at MWG Operon
  13. Sequencing at Agencourt Bioscience
  14. Sequencing at CUGI
  15. Analyzing Sequences with ApE
  16. Using Apes (A Plasmid Editor)
  17. VeriPart for DNA sequences of Registry Parts
  18. The Optimus for optimizing codons
  19. Wiser Optimizer Not working right now
  20. GGAJET Junction Deign Tool
  21. GGA Primer Pairs Designed for You


Bio113 Lab Protocols

General Lab Resources

  1. How to Keep a Lab Notebook
  2. Open Access Libraries


Discovering New Promoters with Synthetic Biology

  1. pClone Basic receiving plasmid for GGA
  2. pClone Green receiving plasmid for GGA
  3. understanding GGA and removal of transcriptional terminator (TT)
  4. Golden Gate Assembly for Bio113
  5. Building dsDNA with Oligos
  6. Promega Tm Calculator
  7. The Loligator
  8. Ligation Protocol
  9. Zippy Transformation
  10. Synergy Machine Protocol
  11. Colony PCR to verify successful GGA
  12. Optimize your Gel
  13. Pouring an agarose gel
  14. Calculate MWs
  15. 1kb Plus MW markers
  16. Ethanol Precipitate to clean DNA (alternative protocol to spin column)
  17. Registry of Standardized DNA Parts hosted by iGEM
  18. Registry of Functional Promoters hosted at Davidson College


TAS2R38 Allele Testing

  1. isolate genomic DNA from a single hair follicle
  2. TAS2R38 PCR amplification
  3. Setting up PCR mixtures
  4. Promega Tm Calculator
  5. PCR and Mg2+ concentration
  6. Prepare PCR product for sequencing (for Bio113 lab use)
  7. Sequencing at MWG Operon
  8. Analyzing Sequences with ApE
  9. Using Apes (A Plasmid Editor)


Evolution of Antibiotic Resistance

  1. glycerolstocks How to Make Glycerol Stocks of Bacteria
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