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This protocol is for extracting DNA from TRIZOL preparations after the RNA-containing upper phase has been completely removed. It is based on the online protocol by Shirley Zhu of the Chang Lab (http://changlab.stanford.edu/DNAextractionfromTRIZOL_Organicphase_update).
== '''Initial sample preparation''' ==#* Perform TRIZOL extraction as you normally would for RNA extraction, ie by mechanically disrupting tissues in Qialyser. Approximately 20 mg of tissue is sufficient. #* After removing the RNA-containing aqueous (upper) phase, re-centrifuge tubes (now containing only the interphase and organic (lower) phase) at 12,000 G for 5 min at 4 deg C. #* Remove any remaining upper phase, taking care to remove everything without damaging the interphase. Samples can now be stored at 4 deg C for days to a couple of weeks.
==Materials==
* Back Extraction Buffer: Containing 4 M Guanidine Thiocyanate (FW 118.16), 50 mM Sodium Citrate NaCl (FW 294.1) and 1 M Tris base (FW 121.14), prepared in ddH2O and sterile filtered. For 25 mL, combine 11.82 g Guanidine Thiocyanate, 0.37 g Sodium Citrate NaCl and 3.03 g Tris base. Be careful to add small volumes of water, gradually, as the reagents will come up to the required volume VERY quickly.
* Isopropanol
* 70% Ethanol
* Qiagen Elution Buffer (or similar TE buffer)
#Centrifuge tubes at 12,000 G for 30 min at room temperature.
##Note: After this step is complete, set the centrifuge to cool to 4 deg C.
#Dissolve DNA pellet in 50 uL of Qiagen elution buffer. Samples can now be stored at 4 deg C.
##Note: Additional washing steps (using phenol, chloroform and isoamylalcohol) should be performed if a more pure pellet is required, however, this preparation should be sufficient for running qPCR/copy number experiments.
[[ Category: Molecular Biology ]]
[[ Category: Mouse Tissues ]]
[[ Category: Mitochondria ]]
[[ Category: DNA Purification ]]
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