Recipes & Protocols

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(LTE Assays)
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*[ IDT OligoAnalyzer]
*[ IDT OligoAnalyzer]
*[ Reverse complement]
*[ Reverse complement]
*[ Genewiz Sequencing]
*[ GenoBase Keio]
*[ GenoBase Keio]
*[ DNA Restriction Site Analyzer]
*[ DNA Restriction Site Analyzer]
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*[ Green RNA to CT of AB]
*[ Green RNA to CT of AB]
*[ mirVana mRNA Isolation Kit]
*[ mirVana mRNA Isolation Kit]
*A helpful [ tutorial] by Margaret Hunt
*[ Dunham lab] has some very helpful tips on microarray & genotyping studies

Revision as of 16:13, 9 July 2013



Gene/Protein Info


Primer designing for gene expression profiling (bacteria)

➢Get the gene name (eg: MYD88)

  • Go to NCBI (, 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 ( 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 (
  • 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 (

Primers for Kan


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


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