Extraction of DNA from TRIZOL preparations - Revision history

Davebrid: added categories, removed table of contents

2015-09-25T17:42:12Z

added categories, removed table of contents

← Older revision		Revision as of 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).		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.		#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.		##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.		#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.		##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__

Erinstephenson at 20:49, 19 February 2015

2015-02-19T20:49:55Z

← Older revision		Revision as of 20:49, 19 February 2015
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	== '''DNA extraction''' ==		== '''DNA extraction''' ==
	=== You will need: ===		=== You will need: ===
	====Back Extraction Buffer====		==== 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.		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.		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====		==== 2. Isopropanol====
	====70% Ethanol====		==== 3. 70% Ethanol====
	====Qiagen Elution Buffer (or similar TE buffer)====		==== 4. Qiagen Elution Buffer (or similar TE buffer)====

	=== Extraction steps ===		=== Extraction steps ===

Erinstephenson: /* Extraction steps */

2015-02-19T20:48:57Z

Extraction steps

← Older revision		Revision as of 20:48, 19 February 2015
Line 18:		Line 18:

	=== Extraction steps ===		=== 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.		#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.		#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.		##Note: After this step is complete, set the centrifuge to cool to 4 deg C.

Erinstephenson: This protocol is for extracting DNA from TRIZOL preparations after the RNA-containing upper phase has been completely removed

2015-02-19T20:46:30Z

This protocol is for extracting DNA from TRIZOL preparations after the RNA-containing upper phase has been completely removed

New page

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.