User:Anthony Salvagno/Notebook/Research/2009/09/28/Ligation with pBR322

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Preloading Nanodrops

  • Short strand of pBR - 1.8kb, 23ng/ul, 19nM
  • long strand of pBR - 2.5kb, 34ng/ul, 20nM
  • T+B - 136.4ng/ul, ~10uM
  • Anchor - 1.1kb, 286.3ng/ul, 390nM

Ligation Setup

{{#widget:Google Spreadsheet |key=t7toion92H2MsSGo3gQmIxQ |width=800 |height=300 }}

  • With 4nM additions (of T+B) every 30 min
  • T+B gets 3 additions
  • Total rxn time = 2 hours

Results

Succesful?
Capture 00021.JPG
If you look really carefully, you can see a band in between the two. Now that band may be unligated plasmid. I don't really know what else it could be. The bottom band is definitely 1.1kb anchor. The top band could very well be ligation product. It is inconclusive because in my haste I did not run a ladder. But I will gel extract and test anyways. Let's hope upon hopes. I will also run the ligation tomorrow with the rest of my digested pBR322 (20ul left).