Difference between revisions of "Extraction of DNA from TRIZOL preparations"
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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== |
| − | + | * 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) | ||
| − | + | == 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. | |
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| − | #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. | ||
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| + | [[ Category: Molecular Biology ]] | ||
| + | [[ Category: Mouse Tissues ]] | ||
| + | [[ Category: Mitochondria ]] | ||
| + | [[ Category: DNA Purification ]] | ||
| + | |||
| + | __NOTOC__ | ||
Latest revision as of 17:42, 25 September 2015
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)
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.
- 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.
