Preparation of 3M sodium acetate (50ml):
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3M sodium acetate is used to precipitate DNA or RNA from ethanol.
For 50 ml,
20.412 g Sodium acetate [ CH3COONa•3H2O, trihydrate]
~6 ml acetic acid to pH, adjust the pH to 5.2 using Glacial acetic acid.
add dH2O to make 50 ml.
Friday, October 17, 2008
Monday, October 6, 2008
DNA /RNA purification
After lysing the samples. DNA or RNA needs to be purified. If it is RNA sample the lysis buffer must contain something (2-merceptoethanol) that will destroy all RNAse enzyme activity.
Then you can purify samples in two ways,
a) Phenol-chloroform extraction,
b) Using kit column,
a) Phenol-chloroform extraction:
Materials:
Saturated phenol, chloroform, 3 M sodium acetate pH 5.2 or 5 M ammonium acetate, 100% ethanol or isopropanol.
Methods:
1. Add equal volume of saturated phenol:chloroform (1:1) to the DNA solution.
2. Mix well. Most DNA solutions can be vortexed for 10 sec. If the molecular weight of the DNA is high (eukaryotic genomic DNA) then sample should be gently rocked.
3. Spin in a microfuge for 3 min.
4. Carefully collect the supernatant layer to a new tube, being careful to avoid the interface. (repeat Steps 1-4 until an interface is no longer visible).
5. To remove traces of phenol, add an equal volume of chloroform to the aqueous layer.
6. Spin in a microfuge for 3 min.
7. Remove aqueous layer to new tube.
8. Measure the volume of the DNA sample. add 1/10 volume of sodium acetate, (final concentration of 0.3 M) or an equal volume of 5 M ammonium acetate (final concentration of 2.0-2.5 M). Mix well.
9. Add 2.5 volumes of cold 100% ethanol or isopropanol. Mix well.
10.Place on ice or at -20 degrees C for >20 minutes.
11.Spin a maximum speed in a microfuge 10-15 min. Carefully remove supernatant.
12.Add 1 ml 70% ethanol. Mix. Spin briefly. Carefully remove supernatant.
13.Air dry or briefly vacuum dry pellet.
14.Resuspend pellet in the appropriate volume of TE or water.
b) Using kit centrifuge column:
Materials:
Appropriate kit from Qiagen or Zymoresearch etc.
Methods:
Most of the company use the following common method to purify DNA.
1. Add 2 volumes of DNA binding solution. Mix it.
2. Transfer the mix to the column on a 2ml collection tube.
3. Spin at 10,000rpm for 10-30 sec. All the DNA will bind to the column.
4. Discard the solution in the collection tube and reuse the tube.
5. Add almost 200ul of washing solution containing 100% ethanol to the column.
6. Spin at 10,000rpm for 10-30 sec. Discard flow through column.
7. Repeat steps 5 and 6.
8. spin at 13,000rpm for 1 min to remove all ethanol residue.
9. Transfer the column to a 1ml eppendorf. add appropriate folume of TE or water directly on the column.
10.Spin for 2 min at high speed.
11.Store the DNA sample at -20 degree centrigade.
See also:
http://userpages.umbc.edu/~jwolf/m4.htm
http://en.wikipedia.org/wiki/Guanidinium_thiocyanate-phenol-chloroform_extraction
Then you can purify samples in two ways,
a) Phenol-chloroform extraction,
b) Using kit column,
a) Phenol-chloroform extraction:
Materials:
Saturated phenol, chloroform, 3 M sodium acetate pH 5.2 or 5 M ammonium acetate, 100% ethanol or isopropanol.
Methods:
1. Add equal volume of saturated phenol:chloroform (1:1) to the DNA solution.
2. Mix well. Most DNA solutions can be vortexed for 10 sec. If the molecular weight of the DNA is high (eukaryotic genomic DNA) then sample should be gently rocked.
3. Spin in a microfuge for 3 min.
4. Carefully collect the supernatant layer to a new tube, being careful to avoid the interface. (repeat Steps 1-4 until an interface is no longer visible).
5. To remove traces of phenol, add an equal volume of chloroform to the aqueous layer.
6. Spin in a microfuge for 3 min.
7. Remove aqueous layer to new tube.
8. Measure the volume of the DNA sample. add 1/10 volume of sodium acetate, (final concentration of 0.3 M) or an equal volume of 5 M ammonium acetate (final concentration of 2.0-2.5 M). Mix well.
9. Add 2.5 volumes of cold 100% ethanol or isopropanol. Mix well.
10.Place on ice or at -20 degrees C for >20 minutes.
11.Spin a maximum speed in a microfuge 10-15 min. Carefully remove supernatant.
12.Add 1 ml 70% ethanol. Mix. Spin briefly. Carefully remove supernatant.
13.Air dry or briefly vacuum dry pellet.
14.Resuspend pellet in the appropriate volume of TE or water.
b) Using kit centrifuge column:
Materials:
Appropriate kit from Qiagen or Zymoresearch etc.
Methods:
Most of the company use the following common method to purify DNA.
1. Add 2 volumes of DNA binding solution. Mix it.
2. Transfer the mix to the column on a 2ml collection tube.
3. Spin at 10,000rpm for 10-30 sec. All the DNA will bind to the column.
4. Discard the solution in the collection tube and reuse the tube.
5. Add almost 200ul of washing solution containing 100% ethanol to the column.
6. Spin at 10,000rpm for 10-30 sec. Discard flow through column.
7. Repeat steps 5 and 6.
8. spin at 13,000rpm for 1 min to remove all ethanol residue.
9. Transfer the column to a 1ml eppendorf. add appropriate folume of TE or water directly on the column.
10.Spin for 2 min at high speed.
11.Store the DNA sample at -20 degree centrigade.
See also:
http://userpages.umbc.edu/~jwolf/m4.htm
http://en.wikipedia.org/wiki/Guanidinium_thiocyanate-phenol-chloroform_extraction
Wednesday, October 1, 2008
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