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The University of Aberdeen Rowett Institute of Nutrition and Health
Isolation of DNA from Tri-reagent TRI-reagent
Sample preparation
Dr Jennifer Duff and Dr Alison Richmond
Post docs
[email protected]
16 April 2009
When citing this SOP you should acknowledge both NuGO and the appropriate NuGO partner
institution that has made the SOP available. Please use a form of words such as:
We used the NuGO Standard Operating Procedure (SOP) number 52 produced by the University
of Copenhagen. Details of the SOP are available via the web link:
http://www.nugo.org/frames.asp?actionID=38662&action=loginFromPP
INTRODUCTION
TRI Reagent is a complete and ready-to-use reagent for isolation of total RNA
or the simultaneous isolation of RNA, DNA and proteins from samples of
human, animal, plant, yeast, bacterial and viral origin. The technique performs
well with small and large quantities of tissue or cultured cells, and allows
simultaneously processing of a large number of samples. Refer to
Manufacturers protocol
REAGENTS/EQUIPMENT
RNase/DNase free 2.0/1.5/0.5 ml eppendorf tubes
Rnase/DNase free pipette tips of appropriate size
TRI-reagent (Sigma, T-9424) – protect from light, store 40C
Ethanol – 100% and 75 %
Refrigerated Centrifuge (up to 12,000 x g)
Ice bucket
Gloves
Eye protection
Fume hood
Timer
Isolation of total RNA with TRI-reagent
Draft SOP RRI-NuGO-01
Wash Buffer
0.1 M sodium citrate in 10 % ethanol
Solubilisation buffer
8 mM NaOH
Guideline
RRI-NuGO-03
The University of Aberdeen Rowett Institute of Nutrition and Health
PROCEDURE
1. Follow procedure for RNA isolation (RRI-NuGO/01) until step 7.
Interphase/phenol phase can be stored overnight or few days at 4 C if
necessary. For longer, store at –80oC. When aquaeous phase (RNA
containing) is completely removed add 0.3 ml (per 1 ml TRI) of 100%
ethanol to interphase / phenol phase then mix by inversion.
2. Store the samples at room temperature (RT) for 2 – 3 minutes then
sediment the DNA at 2000 g for 5 minutes at 4C.
3. Remove and, if required for protein isolation, keep the phenol ethanol
supernatant and store at 4oC.
4. Wash the remaining pellet twice in a solution containing 0.1 M sodium
citrate in 10 % ethanol (use 1 ml of solution per 1 ml initial TRI reagent).
For each wash store pellet for 30 minutes in wash solution at RT with
periodic mixing followed by centrifugation at 2,000 g for 5 minutes at 4C.
5. Suspend (flick to dislodge) the DNA in 75 % ethanol (1.5 – 2 ml of ethanol
per 1 ml of TRI) and store for 10 – 20 minutes at RT with periodic mixing.
Samples stored in 75 % ethanol can be kept at 4C for months
6. Centrifuge at 2,000 g for 5 minutes at 4C, this removes pinkish colour
from DNA pellet.
7. Remove the ethanol wash and briefly air dry the pellet by keeping tubes
open for 3 – 5 minutes at RT.
8. Resuspend the DNA pellet in 8M NaOH. Typically add 0.3-0.6 ml 8M
NaOH to DNA isolated from 50-70 mg tissue. DNA can be centrifuged if
believed to contain insoluble material at 12,000 g for 10 mins and transfer
supernatant to new tube. High viscosity indicates presence of high
molecular weight DNA.
9. Samples solubilsed in NaOH can be stored overnight at 4 C. For
prolonged storage adjust pH to 7 – 8 and then supplement with EDTA.
Guideline
RRI-NuGO-03