User:Anthony Salvagno/Notebook/Research/2011/03/04/Prepping Unzipping With pALS

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PCR

I cleaned up the PCR reaction from Thursday and the nanodrop revealed:

  • 227ng/ul --> 85nM in 30ul (total of 6.8ug of DNA)

BstXI Digestion

{{#widget:Google Spreadsheet |key=0Agbdciapt4QZdFRQdlV3cGV5eHgzTEE2VnZGRGlua3c |width=500 |height=300 }}

Adapter Annealing

Since I got a new bottom oligo with 5' biotinylation I will have to anneal a new adapter duplex. {{#widget:Google Spreadsheet |key=0Agbdciapt4QZdGVoNElGZ0piZ0FzR0hGOFdBWkhZdGc |width=500 |height=300 }}

Ligation

I only have SappBR so I will need to ligate the adapter to here first and then ligate this to the digested pALS.

SappBR-Adapter Ligation

{{#widget:Google Spreadsheet |key=0Agbdciapt4QZdHZpSjF5UThUek5JN3NtR1RtUkFoenc |width=500 |height=300 }}

After purification:

  • ApBR:
    • 89ng/ul --> ~31nM -- ~2.7ug
  • DpALS:
    • 151.1ng/ul --> ~57nM -- ~4.5ng

ApBR-DpALS Ligation

{{#widget:Google Spreadsheet |key=0Agbdciapt4QZdDcyZ2JEanhRS3FRTUNqc2luMUM1Unc |width=500 |height=300 }}