Extraction of DNA from TRIZOL preparations: Difference between revisions

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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~~'''~~ ==

== Initial sample preparation==

#Perform TRIZOL extraction as you normally would for RNA extraction.

* 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.

* 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.

* 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)

== Extraction steps ==

~~== '''DNA extraction''' ==~~

#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.

~~=== 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.

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#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 ]]

__NOTOC__

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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''' ==
+== Initial sample preparation==
-#Perform TRIZOL extraction as you normally would for RNA extraction.
+* 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.
+* 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.
+* 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)
+== Extraction steps ==
-== '''DNA extraction''' ==
+#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.
-=== 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.
@@ Line 28: / Line 23: @@
 #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 ]]
+__NOTOC__