Dna Rna Isolation Trizol
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DNA Isolation: TRIZOL Reagent 1. Tissues: KEEP FROZEN on dry ice, Homogenize in 1 mL Trizol, under Fume Hood using tissue homogenizer. a) Put probe on drill. b) Set up epi tubes in rack. c) Dislodge tissue form cryotube with forceps, tap into homogenizer tube, throw out cryotube. d) As soon as you add Trizol, homogenize from low to high speed for one min. e) Pipette into epi tube, discard probe and homogenizer tube (Can freeze at -20C) (Thaw on bench, at room temp to continue) 2. Phase Separation : a) Incubate cells/TRIZOL at 15-30°C for 5 minutes. b) Add 200 ul of chloroform per 1 ml of TRIZOL used. Cap tubes and vortex for 15 seconds. c) Incubate at 15-30°C (room temp) for 15 minutes. d) Centrifuge samples at 12,000 RPM for 15 minutes at 2-8°C. (Cold room) *after centrifugation: aqueous = RNA. interphase = protein/DNA. organic = DNA. 3. DNA Precipitation : a) Remove aqueous (RNA) phase to waste, or save in cryovial. b) Add 300 ul 100% Ethanol (200 proof EtOH) per 1 ml TRIZOL used to interphase/organic phase. c) Vortex gently. d) Incubate samples at 15-30°C for 2-3 minutes. e) Centrifuge at 12,000 RPM for 5 minutes at room temp. 4. DNA Wash : a) Remove phenol/ethanol sup (contains protein) to waste. (Pipet into 50ml conical & dispose in Trizol Waste container) b) Wash DNA pellet two times in 0.1M sodium citrate solution. Add 1 ml Na citrate per 1 ml Trizol used. c) Gently vortex. Incubate samples in wash solution at 15-30°C for 30 minutes (periodic mixing). d) Spin at 12,000 RPM for 5 minutes at room temp. e) After washes resuspend DNA in 1 ml 75% Ethanol per 1 ml TRIZOL used. USE: 200 proof EtOH and PCR- grade water. (eg. For 50ml, 37.5ml of ethanol and 12.5ml of water) f) Incubate at 15-30°C for 10-20 minutes (periodic mixing). g) Centrifuge at 12,000 RPM for 5 minutes at room temp. Remove sup using p200 pipettor. h) Briefly dry pellet for 2-5 minutes under vacuum. (Can leave on bench) *Add 100μl of water and freeze at –80 or continue to next step. 5. Redissolving the DNA : a) Dissolve pellet in 100ul (or 200-500ul for thymus) of sterile water with a pipette. Can place sample at 55°C for 10 minutes to increase solubility. b) *Solution may still contain insoluble fragments (membrane, protein, etc). c) Centrifuge samples at 12,000 RPM for 10 minutes in cold room to remove insoluble material. d) Transfer sup containing DNA to a new, autoclaved, labeled, flip-top eppendorf. e) Spec. AMOUNT: 1ul of sample + 50ul of dH2O (198ul H2O+2ul sample) f) Store at -80. Solutions : TRIZOL Reagent Life Technologies #15596 Ethanol : 100% & 70% 0.1M sodium citrate in 10% Ethanol : 8mM NaOH : 2.941 g sodium citrate .032 g NaOH 90 ml H 2 O 100 ml H 2 O 10 ml 100 % Ethanol
description
Sorbitol, also known as glucitol, is a sugar alcohol, which the human body metabolizes slowly. It can be obtained by reduction of glucose, changing the aldehyde group to a hydroxyl group. Sorbitol is found in apples, pears, peaches, and prunes.[1] It is synthesized by sorbitol-6-phosphate dehydrogenase, and converted to fructose by succinate dehydrogenase and sorbitol dehydrogenase.[1] Succinate dehydrogenase is an enzyme complex that participates in the citric acid cycle.[1]
Transcript of Dna Rna Isolation Trizol
DNA Isolation: TRIZOL Reagent 1. Tissues: KEEP FROZEN on dry ice, Homogenize in 1 mL Trizol, under Fume Hood
using tissue homogenizer. a) Put probe on drill. b) Set up epi tubes in rack. c) Dislodge tissue form cryotube with forceps, tap into homogenizer tube, throw out cryotube. d) As soon as you add Trizol, homogenize from low to high speed for one min. e) Pipette into epi tube, discard probe and homogenizer tube (Can freeze at -20C) (Thaw on bench, at room temp
to continue)
2. Phase Separation: a) Incubate cells/TRIZOL at 15-30°C for 5 minutes. b) Add 200 ul of chloroform per 1 ml of TRIZOL used. Cap tubes and vortex for 15 seconds. c) Incubate at 15-30°C (room temp) for 15 minutes. d) Centrifuge samples at 12,000 RPM for 15 minutes at 2-8°C. (Cold room)
*after centrifugation: aqueous = RNA. interphase = protein/DNA. organic = DNA. 3. DNA Precipitation: a) Remove aqueous (RNA) phase to waste, or save in cryovial. b) Add 300 ul 100% Ethanol (200 proof EtOH) per 1 ml TRIZOL used to interphase/organic phase. c) Vortex gently. d) Incubate samples at 15-30°C for 2-3 minutes. e) Centrifuge at 12,000 RPM for 5 minutes at room temp.
4. DNA Wash: a) Remove phenol/ethanol sup (contains protein) to waste. (Pipet into 50ml conical & dispose in Trizol Waste
container) b) Wash DNA pellet two times in 0.1M sodium citrate solution. Add 1 ml Na citrate per 1 ml Trizol used. c) Gently vortex. Incubate samples in wash solution at 15-30°C for 30 minutes (periodic mixing). d) Spin at 12,000 RPM for 5 minutes at room temp. e) After washes resuspend DNA in 1 ml 75% Ethanol per 1 ml TRIZOL used. USE: 200 proof EtOH and PCR-
grade water. (eg. For 50ml, 37.5ml of ethanol and 12.5ml of water) f) Incubate at 15-30°C for 10-20 minutes (periodic mixing). g) Centrifuge at 12,000 RPM for 5 minutes at room temp. Remove sup using p200 pipettor. h) Briefly dry pellet for 2-5 minutes under vacuum. (Can leave on bench)
*Add 100µl of water and freeze at –80 or continue to next step.
5. Redissolving the DNA: a) Dissolve pellet in 100ul (or 200-500ul for thymus) of sterile water with a pipette. Can place sample at
55°C for 10 minutes to increase solubility. b) *Solution may still contain insoluble fragments (membrane, protein, etc). c) Centrifuge samples at 12,000 RPM for 10 minutes in cold room to remove insoluble material. d) Transfer sup containing DNA to a new, autoclaved, labeled, flip-top eppendorf. e) Spec. AMOUNT: 1ul of sample + 50ul of dH2O (198ul H2O+2ul sample) f) Store at -80.
Solutions: TRIZOL Reagent Life Technologies #15596 Ethanol: 100% & 70% 0.1M sodium citrate in 10% Ethanol: 8mM NaOH: 2.941 g sodium citrate .032 g NaOH 90 ml H2O 100 ml H2O 10 ml 100 % Ethanol
RNA Isolation: TRIZOL Reagent
*If frozen, thaw quickly in 37°C bath, transfer to 15ml conical w/ 10ml of cold 1xPBS to wash freezing media.
1. Homogenization: Cells: Pellet at 1500 for 5 min. Aspirate dry. Add 1 ml of room temp TRIZOL (do not take directly from stock) per 5-10x106 cells. Lyse cells by repetitive pipetting. Tissues: Homogenize in 1 mL Trizol using tissue homogenizer (Omni International).
2. Phase Separation: -Incubate cells/TRIZOL at 15-30°C for 5 minutes.
-Add 200 ul of chloroform per 1 ml of TRIZOL used. Cap tubes and vortex for 15 seconds. -Incubate at 15-30°C for 10-15 minutes w/ a vortex half way through. -Centrifuge samples at 12,000 RPM for 15 minutes at 2-8°C. (cold room)
*after centrifugation: aqueous = RNA. interphase = protein/DNA. organic = DNA. 3. RNA Precipitation:
-Transfer aqueous phase to an eppendorf tube CAREFULLY, using a P-200 pipettor. Discard lower, DNA phase or freeze at -80. -Add 500 ul Isopropyl Alcohol, mix gently, and incubate at room temp for 10 minutes. -Centrifuge at 12,000 X g for 10 minutes at 4°C. The RNA precipitate will form a gel-like pellet at the bottom of the tube.
4. RNA Wash: -CAREFULLY pull off supernatant with a P-200.
-Wash the pellet once with 1ml of 75% Ethanol (ICE COLD). STOP HERE IF NO SPEC AT THIS TIME. FREEZE AT -80C.
-Centrifuge at 12,000 X g for 15 minutes at 4°C. The RNA should form a visible, white pellet. 5. Dissolving RNA: -CAREFULLY pull off supernatant with a P-200. -Air dry pellet (do not dry under vacuum). Allow to dry completely so there is no residual ethanol.
-Dissolve RNA in Rnase-free water (DEPC WATER), volume dependent on size of tissue, number of cells, or size of pellet. 10-100ul -Check RNA concentration by spec.
Solutions: TRIZOL Reagent Life Technologies #15596 Ethanol: 100% & 75% Isopropyl Chloroform
