Recipes & Protocols

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(Site-Directed Mutagenesis)
(Site-Directed Mutagenesis)
Line 49: Line 49:
*[ QuikChange II STM Kit Protocol]
*[ QuikChange II STM Kit Protocol]
*[ QuikChange Primer Design (Agilent)]
*[ QuikChange Primer Design (Agilent)]
===Protein Expression and Purification===
===Protein Expression and Purification===
*[ BugBuster Protein Extraction Protocol]
*[ BugBuster Protein Extraction Protocol]

Revision as of 14:04, 12 June 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

Protein Expression and Purification

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