Phenol:Chloroform Extractions and EtOH Precipitations - MspI Digestions of C.virginica DNA from Earlier Today

2018
Crassostrea virginica
Eastern oyster
EtOH precipitation
phenol-chloroform cleanup
Author

Sam White

Published

January 11, 2018

The two MspI restriction digestions from earlier today for our project with Qiagen were subjected to phenol:chloroform cleanup and subsequent ethanol precipitations.

Phenol:chloroform clean up procedure:

  1. Added equal volume (50μL) of phenol:chloroform:IAA (25:24:1) to each sample.

  2. Vortexed.

  3. Centrifuged 5mins, 16,000g at room temperature.

  4. Transferred aqueous phase (top layer) to clean 0.5mL snap-cap PCR tube.

  5. Added equal volume of chloroform (50μL) to aqueous phase.

  6. Vortexed.

  7. Centrifuged 5mins, 16,000g at room temperature.

  8. Transferred aqueous phase (top layer) to clean 0.5mL snap-cap PCR tube.

Performed ethanol precipitation on both samples according to lab protocol.

Resuspended precipitated DNA in 25μL Buffer EB (Qiagen).

Will quantify with Qubit 3.0.