To remove ethidium bromide from CsCl-gradient purified DNA, repeated extraction with organic solvents is commonly used. Subsequently, CsCl can be removed by dialysis or precipitation.Source: "Guide to Molecular Cloning, Third Edition", translated by P.T. Huang et al.
Operation method
Removal of ethidium bromide from DNA by organic solvent extraction experiment
Principle
To remove ethidium bromide from CsCl gradient-purified DNA, repeated extraction with organic solvents is commonly used. CsCl can then be removed by dialysis or precipitation.
Materials and Instruments
DNA Sample Move I. Materials For more product details, please visit Aladdin Scientific website.
Ethanol Water-saturated isoamyl alcohol or n-butanol Phenol TE
Dialysis tubing and clips Centrifuge and rotary head
1. Buffers and solutions
Ethanol, water-saturated isoamyl alcohol or n-butanol (to remove ethidium bromide from DNA prepared by centrifugation want one of the above two organic solvents.) Phenol, phenol: chloroform (1:1, V/V), TE ( pH 8.0).
2. Nucleic acids and oligonucleotides
DNA samples purified by gradient centrifugation with CsCl
3. centrifuges and rotors
Sorvall RT-6000 centrifuge (or equivalent) with HL-4 rotor head and 50 ml tubes
Sorvall SS-34 head (or equivalent)
4. Specialized equipment
Dialysis tubing and clips or microconcentrator for rotary dialysis (Amicon) .
Methods
1. Extraction of DNA solution, removal of ethidium bromide
(1) Add DNA solution to a glass or plastic tube, add an equal volume of water-saturated n-butanol or isoamyl alcohol, and cap the tube tightly.
(2) Vibrate to mix the organic and aqueous phases.
(3) Centrifuge the mixture at room temperature for 3 min at 450 g (1500 r/min on a Sorvall RT-6000 centrifuge with an HL-4 turntable and a 50 ml centrifuge tube) or leave at room temperature until the organic and aqueous phases separate.
(4) Transfer the upper organic phase (which is a nice deep pink color) to an appropriate waste container using a Pasteur pipette.
(5) Repeat the extraction (steps 1 to 4) 4 to 6 times until both the aqueous and organic phases are no longer pink.
2. Removal of CsCI from DNA solution
(1) Remove CsCl from DNA solution by ethanol precipitation (steps 7-12), or by rotary dialysis using a microconcentrator (Ami-con) or overnight dialysis with 2L of TE (pH 8.0) for 16 h (with constant buffer changes). If one of the latter two methods is used, proceed directly to step 8.
(2) To precipitate DNA from CsCl-DNA solution, add 3 times the volume of CsCI solution and mix thoroughly.
This additional step dilutes the CsCl and prevents it from being precipitated by ethanol.
(3) Add 8 times the volume of ethanol (1 volume of CsCl-DNA solution before dilution with water in step 2) to the DNA solution and mix thoroughly, store at 4°C for at least 15 min.
Higher DNA recoveries are obtained if the precipitation reaction is left at 4°C overnight.
Important: CsCl will precipitate if the ethanol solution of DNA is stored at -20 °C.
(4) Collect the DNA precipitate by centrifugation at 20,000 g for 15 min at 4°C (Sorvall SS-34 turntable 13000 r/min).
(5) Pipette the supernatant into a clean centrifuge tube and add an equal volume of ethanol solution. Place the mixture at 4°C for at least 15 min, then centrifuge at 20,000 g for 15 min (Sorvall SS-34 head) to collect the DNA precipitate.
(6) Wash the DNA precipitate obtained twice above with 70% ethanol, then remove as much of the 70% ethanol as possible and allow the residual liquid to evaporate at room temperature.
(7) Dissolve the DNA precipitate with 2 ml H2O or TE (pH 8.0).
For DNA sequencing, the DNA should be dissolved in H2O; if the DNA is to be stored for a long time, TE (pH 8.0) is a better choice.
(8) If it is clear that this re-solubilized DNA still contains ethidium bromide, as judged by its color or fluorescence when exposed to ultraviolet light, extract it again with phenol or phenol:chloroform, and precipitate the DNA with ethanol.
(9) Measure the OD260 value of the final DNA solution and calculate the concentration of DNA. Store at -20°C in small portions.