User:Kristina M Holton/Notebook 2007 7 2

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