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Extraction of DNA from TRIZOL preparations

164 bytes added, 17:42, 25 September 2015
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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)
 == '''DNA extraction''' ===== You will need: ======= 1. Back Extraction Buffer====Containing 4 M Guanidine Thiocyanate (FW 118.16), 50 mM Sodium Citrate NaCi (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 NaCi 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.==== 2. Isopropanol======== 3. 70% Ethanol======== 4. Qiagen Elution Buffer (or similar TE buffer)==== === Extraction steps ===#Add 500 uL of '''Back Extraction Buffer ''' (BEB) for every 1 mL of TRIZOL used in the initial extraction. Place on shaker for 10 min. Always mix by inversion or use the shaker. '''NEVER ''' vortex samples, as it will destroy the DNA.
#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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