DNA Isolation – Ireland Clam Tissue

Performed DNA isolation on clam tissue (sample: S/6/14 #19) supplied by Deborah Cheslett in July 2014 (based on date on letter accompanying the sample).  Sample was preserved in 80% EtOH.  Isolated DNA using the QIAamp Fast DNA Stool Kit (Qiagen), per Carolyn’s request.

Removed tissue from EtOH, blotted dry with Kim Wipes.  Tissue type was not noted in the letter accompanying the sample.

Tissue weighed 117mg, which is just below the recommended range for the the Qiagen stool kit (180 – 220mg is recommended).  Minced tissue with razor blade and processed according to the manufacturer’s protocol for pathogen detection.  Because tissue was very dense/rubbery, the tissue did not lyse during the initial incubation period (95C for 5mins).  Extended this incubation to 2.5hrs in an attempt to lyse the tissue.  Lysis did not fully dissolve the tissue, which was not surprising.

Proceeded with the manufacturer’s protocol and eluted with 100μL of Buffer ATE.

Retained remaining supernatant from the lysis step.

Processed the remaining unlysed tissue using the DNeasy Blood & Tissue Kit (Qiagen) according the manufacturer’s protocol.  Tissue lysis step was performed at 56C O/N.

Sample was eluted with 200μL of Buffer AE.


 

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