Difference between revisions of "Davidson Protocols"
From GcatWiki
Macampbell (talk | contribs) |
Macampbell (talk | contribs) |
||
Line 55: | Line 55: | ||
# 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. | # 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. | ||
# [http://partsregistry.org/AHL List of auto-inducers and their catalog numbers] | # [http://partsregistry.org/AHL List of auto-inducers and their catalog numbers] | ||
− | #[[Synergy Machine Protocol]] | + | # [[Synergy Machine Protocol]] |
'''G. Golden Gate Shuffling''' | '''G. Golden Gate Shuffling''' | ||
− | #[[Media:DNAShuffling.docx]]<br> | + | # [[Media:DNAShuffling.docx]]<br> |
'''H. Computer Tools We Use''' | '''H. Computer Tools We Use''' | ||
# [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Optimize your Gel] | # [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Optimize your Gel] | ||
− | #[http://genedesign.thruhere.net/gd/ Gene Design (Boeke Lab at JHU)] | + | # [http://genedesign.thruhere.net/gd/ Gene Design (Boeke Lab at JHU)] |
# [http://gcat.davidson.edu/iGEM07/genesplitter.html Gene Splitting Web Site] | # [http://gcat.davidson.edu/iGEM07/genesplitter.html Gene Splitting Web Site] | ||
# [http://gcat.davidson.edu/iGEM08/bbprimer.html PCR Primers w/ BioBricks] | # [http://gcat.davidson.edu/iGEM08/bbprimer.html PCR Primers w/ BioBricks] | ||
Line 81: | Line 81: | ||
− | '''I. Bio113 Lab Protocols'' | + | '''I. Bio113 Lab Protocols''' |
− | #[[ | + | # [http://www.bio.davidson.edu/courses/molbio/labnotebook.html How to Keep a Lab Notebook] |
+ | # [http://www.opendoar.org/countrylist.php?cContinent=North%20America#United%20States Open Access Libraries] | ||
+ | # [http://www.bio.davidson.edu/courses/Molbio/Protocols/clean_short.html Ethanol Precipitate to clean DNA (short protocol)] | ||
+ | # [[glycerolstocks How to Make Glycerol Stocks of Bacteria]] | ||
+ | # [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://products.invitrogen.com/ivgn/product/10787018 1kb Plus MW markers] | ||
+ | # [http://www.bio.davidson.edu/courses/Molbio/Protocols/ligation.html Ligation Protocol] | ||
+ | # [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Transformation] | ||
+ | # [[Golden Gate Assembly protocol]] | ||
+ | # [http://www.bio.davidson.edu/courses/Molbio/Protocols/anneal_oligos.html Building dsDNA with Oligos] | ||
+ | # [http://gcat.davidson.edu/SynBio12/ The Loligator] | ||
+ | # [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] | ||
+ | # [[isolate genomic DNA from a single hair follicle]] | ||
+ | # [[Prepare PCR product for sequencing]] after clean and concentration procedure (for Bio113 lab use) | ||
+ | # [http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html Clean and Concentrate DNA with spin column (after PCR, before digestion)] | ||
+ | # [[Golden Gate Assembly protocol]] | ||
+ | # [[TAS2R38 PCR amplification]] | ||
+ | # [[Synergy Machine Protocol]] | ||
+ | # [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Optimize your Gel] | ||
+ | # [http://www.promega.com/biomath/calc11.htm Promega T<sub>m</sub> Calculator] | ||
+ | # [http://gcat.davidson.edu/iGem10/index.html Oligator making dsDNA from oligos] | ||
+ | # [http://gcat.davidson.edu/SynBio12/ The Loligator] | ||
+ | # [[Sequencing at MWG Operon| Sequencing at MWG Operon]] | ||
+ | # [http://gcat.davidson.edu/GcatWiki/images/3/3b/Ape_protocol.pdf Analyzing Sequences with ApE] | ||
+ | # [[Using Apes (A Plasmid Editor)]] |
Revision as of 13:57, 8 August 2012
A. General Lab Information
- How to Keep a Lab Notebook
- Common molecular reagents
- Open Access Libraries
- Standard Assembly
- BioBrick Ends
- Compatibility of Plasmids
- Ethanol Precipitate to clean DNA (short protocol)
- glycerolstocks How to Make Glycerol Stocks of Bacteria
B. Gel Electrophoresis and Purification
- Pouring an agarose gel
- Calculate MWs
- 1kb Plus MW markers
- Macherey-Nagel Gel Purification (improved 260/230 ratios)l
- Qiagen QIAquick Gel Purification
- Qiagen QIAquick Column Regeneration Protocol
- ElectroElute Gel Purification
C. Digestion, Ligation, Transformation
- Digest DNA with restriction enzymes
- Double Digest Guide
- Shrimp Alkaline Phosphatase
- Ligation Protocol
- Choices for Transformation: Heat Shock vs. Zyppy
- Heat Shock Transformation OR Short version of Heat Shock
- Zippy Transformation
- TSS Competent Cell Transformation
- Golden Gate Assembly protocol
D. Minipreps
E. Making New Parts and PCR
- Building dsDNA with Oligos
- The Loligator
- Setting up PCR mixtures
- PCR and Mg2+ concentration
- isolate genomic DNA from a single hair follicle
- Prepare PCR product for sequencing after clean and concentration procedure (for Bio113 lab use)
- Making dsDNA Using Primer Dimers
- Clean and Concentrate DNA with spin column (after PCR, before digestion)
- Colony PCR to Screen for Successful Ligations
- Golden Gate Assembly protocol
- How many clones should I screen?
- TAS2R38 PCR amplification
F. Expression of Phenotypes
- Using degradation tags on proteins such as GFP
- Genomic Insertion Protocol
- 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.
- When inducing with Arabinose, use "2 µL of stock" (10% w/v L-Arabinose) "to every 1 mL" of LB or other liquid.
- 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.
- List of auto-inducers and their catalog numbers
- Synergy Machine Protocol
G. Golden Gate Shuffling
H. Computer Tools We Use
- Optimize your Gel
- Gene Design (Boeke Lab at JHU)
- Gene Splitting Web Site
- PCR Primers w/ BioBricks
- Promega Tm Calculator
- Oligator making dsDNA from oligos
- The Loligator
- How many clones should I screen?
- Lance-olator Oligos for dsDNA assembly old version, we recommend Oligator now
- Access the GCAT-alog of Davidson and MWSU DNA Freezer Stocks
- Sequencing at MWG Operon
- Sequencing at Agencourt Bioscience
- Sequencing at CUGI
- Analyzing Sequences with ApE
- Using Apes (A Plasmid Editor)
- VeriPart for DNA sequences of Registry Parts
- The Optimus for optimizing codons
- Wiser Optimizer Not working right now
I. Bio113 Lab Protocols
- How to Keep a Lab Notebook
- Open Access Libraries
- Ethanol Precipitate to clean DNA (short protocol)
- glycerolstocks How to Make Glycerol Stocks of Bacteria
- Pouring an agarose gel
- Calculate MWs
- 1kb Plus MW markers
- Ligation Protocol
- Zippy Transformation
- Golden Gate Assembly protocol
- Building dsDNA with Oligos
- The Loligator
- Setting up PCR mixtures
- PCR and Mg2+ concentration
- isolate genomic DNA from a single hair follicle
- Prepare PCR product for sequencing after clean and concentration procedure (for Bio113 lab use)
- Clean and Concentrate DNA with spin column (after PCR, before digestion)
- Golden Gate Assembly protocol
- TAS2R38 PCR amplification
- Synergy Machine Protocol
- Optimize your Gel
- Promega Tm Calculator
- Oligator making dsDNA from oligos
- The Loligator
- Sequencing at MWG Operon
- Analyzing Sequences with ApE
- Using Apes (A Plasmid Editor)