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This protocol is for extracting DNA from TRIZOL preparations after the RNA-containing upper phase has been completely removed
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.
#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.
== '''DNA extraction''' ==
=== You will need: ===
====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.
====Isopropanol====
====70% Ethanol====
====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.
#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.
#Transfer aqueous (upper) phase to a clean tube and add 400 uL of ice cold isopropanol (to precipitate the DNA). Mix by inversion. Incubate at room temperature for 5 min.
#Centrifuge tubes at 12,000 G for 15 min at 4 deg C.
#Very carefully remove supernatant (it is very unlikely that the DNA pellet will be visible). Add 500 uL of 70% ethanol and mix by inversion (to wash the pellet- this step removes the salt that co-precipitates with the DNA).
#Centrifuge tubes at 12,000 G for 15 min at 4 deg C.
#Very carefully remove all of the supernatant (once again taking care not to disturb what is probably an invisible DNA pellet).
#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.
== '''Initial sample preparation''' ==
#Perform TRIZOL extraction as you normally would for RNA extraction.
#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.
== '''DNA extraction''' ==
=== You will need: ===
====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.
====Isopropanol====
====70% Ethanol====
====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.
#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.
#Transfer aqueous (upper) phase to a clean tube and add 400 uL of ice cold isopropanol (to precipitate the DNA). Mix by inversion. Incubate at room temperature for 5 min.
#Centrifuge tubes at 12,000 G for 15 min at 4 deg C.
#Very carefully remove supernatant (it is very unlikely that the DNA pellet will be visible). Add 500 uL of 70% ethanol and mix by inversion (to wash the pellet- this step removes the salt that co-precipitates with the DNA).
#Centrifuge tubes at 12,000 G for 15 min at 4 deg C.
#Very carefully remove all of the supernatant (once again taking care not to disturb what is probably an invisible DNA pellet).
#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.