Recipes & Protocols

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(Gibson Assembly)
 
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*[http://ecoli.naist.jp/GB8-dev/index.jsp?page=gene_search.jsp&sf=T GenoBase Keio]
*[http://ecoli.naist.jp/GB8-dev/index.jsp?page=gene_search.jsp&sf=T GenoBase Keio]
*[http://tools.neb.com/NEBcutter2/ DNA Restriction Site Analyzer]
*[http://tools.neb.com/NEBcutter2/ DNA Restriction Site Analyzer]
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*[http://syntheticbiology.org/Tools.html A list of commonly used syntethic biology tools] by SyntheticBiology.org
===Gene/Protein Info===
===Gene/Protein Info===
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===Protocols===
===Protocols===
*[http://openwetware.org/wiki/Choosing_primers_for_qPCR Choosing primers for q-PCR]
*[http://openwetware.org/wiki/Choosing_primers_for_qPCR Choosing primers for q-PCR]
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*[http://openwetware.org/wiki/PCR_Overlap_Extension Soeing PCR - Primer design]
===Primer designing for gene expression profiling (bacteria)===
===Primer designing for gene expression profiling (bacteria)===
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*[http://dunham.gs.washington.edu/protocols.shtml Dunham lab] has some very helpful tips on microarray & genotyping studies
*[http://dunham.gs.washington.edu/protocols.shtml Dunham lab] has some very helpful tips on microarray & genotyping studies
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===Gibson Assembly===
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*[https://www.neb.com/~/media/NebUs/Files/Feature%20Articles/Gibson_Assembly.pdf Introduction to Gibson assembly]
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*[http://2012.igem.org/Team:Freiburg/Project/Golden Golden Gate Cloning] (an alternative) (P: I've never tried this one before, but be my guest)
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*J5 [http://j5.jbei.org/bin/login.pl Primer design tool]
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*Isothermal Assembly (Megason Lab) [https://wiki.med.harvard.edu/SysBio/Megason/IsothermalAssembly]
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*Making aliquots (Megason Lab) [https://wiki.med.harvard.edu/SysBio/Megason/MakingIsothermalAssemblyAliquots]
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Compiled by Betul Kacar and Lily Tran. In the year twothousandandthirteen.
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----
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: Compiled by Betül Kacar, 2013-2014.
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: Team members: Lily Tran, Peter Schnaak and Jen Zhang.
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: Contact betul_AT_gatech_DOT_edu for questions.

Current revision

Contents

Tools

Gene/Protein Info

Protocols

Primer designing for gene expression profiling (bacteria)

➢Get the gene name (eg: MYD88)

  • Go to NCBI (ncbi.nlm.nih.gov), and search in ‘Gene’ for the gene sequence.
  • Type in REL606 (that is the E.coli strain we use, unless stated otherwise) together with the gene name, and get the NM number of the most common transcript variant. (eg: NM_002468.4 for MYD88)
  • Click on the link and go to the gene description page.
  • Scroll down and clink on the link to find the CDS, or the RefSeq.
  • Copy the CDS sequence.
  • Go to the Primer 3 Plus website (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) and paste the CDS sequence in the search box.
  • All the parameters for optimal primers are usually preset. Do not change anything.
  • Click “pick primers’.
  • When the selection of primers comes up, choose a pair that is closest to the 3’ end.
  • Check the primer is 20bp long and the product size is between 150-200bp.
  • Blast to check the specificity of the primer pair (http://blast.ncbi.nlm.nih.gov/)
  • Paste the forward and reverse primer sequences one by one and check if it gives a unique hit. (confirm chromosomal location of the gene from NCBI or other sources)
  • If it gives multiple hits, select a different set of primers from Primer 3 Plus and repeat the same process until a unique set is obtained.

➢ Order primers through Invitrogen:

  • Purification: Desalted
  • Starting Synthesis Scale: 25nmole
  • Ship Medium: Dry
  • Normalization: None
  • Calculate primers to 100uM using DNA suspension Buffer from Teknova cat # T0221

➢ Contact me if you have any questions (betul.kacar@biology.gatech.edu)

Primers for Kan

  • Kan Cassette Test (Fwd): 5’ GTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAG 3’
  • Kan Cassette Test (Rev): 5’ ATTCCGGGGATCCGTCGACCTGCAGTTCGAAGTTCCTATT 3’

Site-Directed Mutagenesis

LTE Assays

araA Marker Test:

  • Make sure to have: HaeII Restriction Enzyme (in the -20C Freezer RE Stocks box), NEB Buffer 4, and good luck
  • araA marker revertant test

RT-PCR

Gibson Assembly



Compiled by Betül Kacar, 2013-2014.
Team members: Lily Tran, Peter Schnaak and Jen Zhang.
Contact betul_AT_gatech_DOT_edu for questions.
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