Difference between revisions of "Davidson Protocols"

From GcatWiki
Jump to: navigation, search
(→‎Bio113 Lab Protocols)
Line 113: Line 113:
 
# [http://www.promega.com/biomath/calc11.htm Promega T<sub>m</sub> Calculator]
 
# [http://www.promega.com/biomath/calc11.htm Promega T<sub>m</sub> Calculator]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/magnesium.html PCR and Mg<sup>2+</sup> concentration]
 
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/magnesium.html PCR and Mg<sup>2+</sup> concentration]
−
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/clean_short.html Ethanol Precipitate to clean DNA (short protocol)]
+
# [[Prepare PCR product for sequencing]] (for Bio113 lab use)
−
# [[Prepare PCR product for sequencing]] after clean and concentration procedure (for Bio113 lab use)
 
 
# [[Sequencing at MWG Operon| Sequencing at MWG Operon]]
 
# [[Sequencing at MWG Operon| Sequencing at MWG Operon]]
 
# [http://gcat.davidson.edu/GcatWiki/images/3/3b/Ape_protocol.pdf Analyzing Sequences with ApE]
 
# [http://gcat.davidson.edu/GcatWiki/images/3/3b/Ape_protocol.pdf Analyzing Sequences with ApE]

Revision as of 21:20, 18 August 2012

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

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

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

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


Bio113 Lab Protocols

General Lab Resources

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


Discovering New Promoters with Synthetic Biology

  1. receiving plasmid for GGA
  2. understanding GGA and removal of transcriptional terminator (TT)
  3. Golden Gate Assembly protocol
  4. Building dsDNA with Oligos
  5. Promega Tm Calculator
  6. The Loligator
  7. Ligation Protocol
  8. Zippy Transformation
  9. Synergy Machine Protocol
  10. Colony PCR to verify successful GGA
  11. Optimize your Gel
  12. Pouring an agarose gel
  13. Calculate MWs
  14. 1kb Plus MW markers
  15. Ethanol Precipitate to clean DNA (alternative protocol to spin column)
  16. Registry of Standardized DNA Parts hosted by iGEM
  17. 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
Retrieved from "https://gcat.davidson.edu/GcatWiki/index.php?title=Davidson_Protocols&oldid=15261"