User:Kristina M Holton/Ovarian Notebook 2007 7 2

From OpenWetWare
Jump to: navigation, search

miRNA Extraction using 1st protocol and Qiagen miRNEasy Kit

  • Large Fibroma -> very very fatty
  • Used 3ml TRIzol from Invitrogen
  • While standing, TRIzol and fat start to pellet at the bottom; removed supernatants and centrifuged both pellet and supernatant
  • After centrifugation, already appeared to be phase separation
  • Still added glycogen + chloroform, stand RT for 10min then centrifuge 10000g 20min
  • Added EtOH: large amount of white, "SDS-like" precipitate
  • Very difficult to add precipitate to RNeasy Mini spin columns, even with additional EtOH

Differences in 2nd protocol (probable fixes):

  • Use 2ml QIAzol for lysis

If: no debris accumulates and the QIAzol remains pink

    • Skip first centrifugation step + removing the supernatant

Else: (Brown QIAzol or debris accumulation)

    • Centrifuge and remove supernatant
  • Try to add all precipitate from EtOH step