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