Preparation of 3M sodium acetate (50ml):
-------------------------------------------
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
Saturday, September 27, 2008
Loading dye or buffer
In gel electrophoresis experiment loading dye or buffer is widely used with DNA and RNA to follow the migration of samples. Typical loading buffer composed of two dyes bromophenol blue and xylene cyanol FF. It can be used for both agarose and polyacrylamide gel. Composition of 6x loading dye or buffer:Recipe no 1:
-----------------------
0.03% bromophenol blue,
10 mM Tris-HCl (pH 7.6),
0.03% xylene cyanol FF,
60% glycerol,
60 mM EDTA.
Recipe no 2:
------------------------------
25mg bromophenol blue (0.25%),
25mg xylene cyanol (0.25%),
4g sucrose (40%),
adjust volume to 10 ml with H2O.
presence of glycerol or sucrose in the solution ensures that the same sample forms a layer at the bottom of the well and the EDTA included in the solution binds divalent metal ions and inhibits metal dependent nucleases.
In 1% agarose gels bromophenol blue co-migrates with ~300 bp DNA and xylene cyanol FF co-migrates with ~4000 bp DNA. Store at +4°C up to 12 months.
In 5 volumes of DNA sample add 1 volume of 6X DNA Loading Dye.
See also,
http://openwetware.org/wiki/Agarose_gel_loading_dye
Tuesday, September 9, 2008
TBE and TE buffer preparation
TBE buffer
TBE buffer is a solution of Tris base, boric acid and EDTA.
This buffer mostly used in DNA electrophoresis experiment.
Recipe for 10x TBE (TrisBorateEDTA) buffer:
--------------------------------------------
Tris base :107g
Boric acid: 55g
0.5M EDTA : 40ml
Add distilled water and adjust the volume to 1 liter.
TE buffer
TE buffer protects DNA or RNA from degradation.
Recipe 1xTE buffer:
--------------------
1ml of 1M Tris-HCL (pH 7.5-8)
0.2ml of 0.5M EDTA (pH 8)
add 988ml of distilled water to make 1 liter 1x TE buffer.
TBE buffer is a solution of Tris base, boric acid and EDTA.
This buffer mostly used in DNA electrophoresis experiment.
Recipe for 10x TBE (TrisBorateEDTA) buffer:
--------------------------------------------
Tris base :107g
Boric acid: 55g
0.5M EDTA : 40ml
Add distilled water and adjust the volume to 1 liter.
TE buffer
TE buffer protects DNA or RNA from degradation.
Recipe 1xTE buffer:
--------------------
1ml of 1M Tris-HCL (pH 7.5-8)
0.2ml of 0.5M EDTA (pH 8)
add 988ml of distilled water to make 1 liter 1x TE buffer.
Sunday, September 7, 2008
What is RNA?
RNA is the short form of ribonucleic acid. Mostly RNAs
are single stranded. sometimes they form double stranded
loop. RNA is composed of four nucleotide bases (A-adenine,
G-guanine, U-uracil and C- cytosine) on a ribose sugar and
phosphate diaester backbone.
There are 3 main difference between DNA and RNA.
1- most of the time RNA is single stranded.
2- RNA contains uracil (U), where as DNA contains thymine (T).
3- RNA backbone composed of ribose sugar and DNA is composed of deoxyrobose sugar.
a- Ribosomal RNA (rRNA)
b- Transfer RNA (tRNA)
c- Messenger RNA (mRNA)
There are other types of RNA such as RNAseP RNA, m1RNA, snRNA, uRNA, siRNA, antisense RNA etc. Among all rRNA is the most abundant RNA of every living cell.
RNA extraction:
DNA is more stable than RNA. At high temperature RNA cannot stay long and ribonuclease enzyme is ubiquitously present in the cell. So, it is difficult to extract RNA from cell. There are seversl ways to extract RNA from samples, the most common of these is Guanidinium thiocyanate-phenol-chloroform extraction. This method often uses a proprietary formulation of this reagent called Trizol. RNA should be stored at at -80°C.
Wear gloves when handling trizol, work RNase-free!
1. Aspirate medium from cells grown in dishes
2. Wash with 1XPBS
3. In the fume hood, add 4ml of Trizol to cells on plastic of 10cm plate 2.5 ml on 60 mm plate,
1 ml on 35mm plate, add 3 ml on 3 D culture ( 35 mm plate)
With a 10ml pipette, pipette up and down for 2-3 min until solution is no longer viscous
4. Transfer onto a 15 ml polypropylene tube
5. Spin down the EHS samples in Sorvall Centrifuge for 10 min at 9000 rpm at 4 °C ( No dissolved protein will be pelleted), and transfer supernatant into fresh tube
6. Add 20% of Chloroform for Trizol ® ( i.e. 0.2 ml Chloroform for each 1 ml Trizol ® )
7. Close tubes firmly and shake and vortex vigorously for at least 15 seconds
8. Let sit at room temperature for 10 minutes.
9. Centrifuge for 15 min at 9000rpm at 4 °C
10. Transfer upper, aqueous phase to fresh tube, avoid transfer of ANY interface!!
11. Add 0.5 volume IPA to the clean supernatant (i.e. 0.5 ml IPA for each ml Trizol) and add 0.5 µl Glycogen for each ml Trizol
12. Mix immediately by inverting tubes 5-8 times
13. Incubate at room, temperature for 5-10 min.
14. Centrifuge at 9000 rpm for 10 min, 4 °C
15. Discard supernatant
16. Washing with 1ml of 75% EtOH (RNase –free) to each tube
17. Resuspend dry pellet in nuclease-free water and keep on ice
EHS samples in 20-30 µl water each
Plastic samples in 60-80
See also,
http://en.wikipedia.org/wiki/RNA
http://www.lbl.gov/lifesciences/BissellLab/labprotocols/rnaextraction.htm
http://www.lebs.cnrs-gif.fr/golinelli/golinelli_eng.htm
http://www.aps.anl.gov/Science/Highlights/2001/ribsome.htm
are single stranded. sometimes they form double stranded
loop. RNA is composed of four nucleotide bases (A-adenine,
G-guanine, U-uracil and C- cytosine) on a ribose sugar and
phosphate diaester backbone.
There are 3 main difference between DNA and RNA.
1- most of the time RNA is single stranded.
2- RNA contains uracil (U), where as DNA contains thymine (T).
3- RNA backbone composed of ribose sugar and DNA is composed of deoxyrobose sugar.
30s rRNA
tRNA
There are three main types of RNA.
tRNAa- Ribosomal RNA (rRNA)
b- Transfer RNA (tRNA)
c- Messenger RNA (mRNA)
There are other types of RNA such as RNAseP RNA, m1RNA, snRNA, uRNA, siRNA, antisense RNA etc. Among all rRNA is the most abundant RNA of every living cell.
RNA extraction:
DNA is more stable than RNA. At high temperature RNA cannot stay long and ribonuclease enzyme is ubiquitously present in the cell. So, it is difficult to extract RNA from cell. There are seversl ways to extract RNA from samples, the most common of these is Guanidinium thiocyanate-phenol-chloroform extraction. This method often uses a proprietary formulation of this reagent called Trizol. RNA should be stored at at -80°C.
Wear gloves when handling trizol, work RNase-free!
1. Aspirate medium from cells grown in dishes
2. Wash with 1XPBS
3. In the fume hood, add 4ml of Trizol to cells on plastic of 10cm plate 2.5 ml on 60 mm plate,
1 ml on 35mm plate, add 3 ml on 3 D culture ( 35 mm plate)
With a 10ml pipette, pipette up and down for 2-3 min until solution is no longer viscous
4. Transfer onto a 15 ml polypropylene tube
5. Spin down the EHS samples in Sorvall Centrifuge for 10 min at 9000 rpm at 4 °C ( No dissolved protein will be pelleted), and transfer supernatant into fresh tube
6. Add 20% of Chloroform for Trizol ® ( i.e. 0.2 ml Chloroform for each 1 ml Trizol ® )
7. Close tubes firmly and shake and vortex vigorously for at least 15 seconds
8. Let sit at room temperature for 10 minutes.
9. Centrifuge for 15 min at 9000rpm at 4 °C
10. Transfer upper, aqueous phase to fresh tube, avoid transfer of ANY interface!!
11. Add 0.5 volume IPA to the clean supernatant (i.e. 0.5 ml IPA for each ml Trizol) and add 0.5 µl Glycogen for each ml Trizol
12. Mix immediately by inverting tubes 5-8 times
13. Incubate at room, temperature for 5-10 min.
14. Centrifuge at 9000 rpm for 10 min, 4 °C
15. Discard supernatant
16. Washing with 1ml of 75% EtOH (RNase –free) to each tube
17. Resuspend dry pellet in nuclease-free water and keep on ice
EHS samples in 20-30 µl water each
Plastic samples in 60-80
See also,
http://en.wikipedia.org/wiki/RNA
http://www.lbl.gov/lifesciences/BissellLab/labprotocols/rnaextraction.htm
http://www.lebs.cnrs-gif.fr/golinelli/golinelli_eng.htm
http://www.aps.anl.gov/Science/Highlights/2001/ribsome.htm
Monday, September 1, 2008
What is DNA? How to extract DNA?
Today I will explain what is DNA and how you can extract it from cell. DNA is the short form of deoxyribonucleic acid. It the hereditary unit of all eukaryotic organisms and most of the prokaryotic ogranisms. DNA is composed of four nucleotides (A- adenine, T- thymine, G- guanine, and C-cytosine) and a backbone made of phospate and dexyribose sugar. In eukaryotic organism Genomic DNA stays inside nucleus. DNA is also present inside mitochondria and plastids organelles. 
DNA has two different ends 5'end and 3' end. DNA is double helix. Two complimentary strand stay together. Adenine (A) always binds with thymine (T) and Guanine (G) always binds with cytosine (C) by hydrogen bonding. In 1962, James D. Watson and Francis Crick received Nobel prize for describing the structure of DNA in Physiology of Medicine.
Basic principles to extract DNA from cells are:
Clean raw cells
Break the cells (lysis) to release DNA into solution
Destroy membrane, proteins and lipids that may bind and destroy the DNA
precipitate DNA by using alcohol
Lets extract DNA from onion.
Materials:
blender, salt, shampoo or soap, tea or coffee filter, ethanol of isopropanol, warm water, tooth pick.
Methods:
clean the onion. Cut into pieces, put into blender, add salt and water. Blend for 3-5 seconds. Filter it using tea or coffee filter. Add 2 or 3 spoon of shampoo. Stir gently so that there will be no bubble. Slowly pour ice cold alcohol into the glass. So there will be two layer. Wait for 3-5 minutes. You will see some white DNA at the surface of the top layer. You can collect this layer by tooth pics and put into another tube with fresh water. Within one minute the DNA will be invisible again in the new tube with water.
If you have questions and if you disagree with anything I have written here just mail me.
See also,
http://library.thinkquest.org/19037/dna_extraction.html
http://en.wikipedia.org/wiki/User:Seans_Potato_Business/DNA
DNA has two different ends 5'end and 3' end. DNA is double helix. Two complimentary strand stay together. Adenine (A) always binds with thymine (T) and Guanine (G) always binds with cytosine (C) by hydrogen bonding. In 1962, James D. Watson and Francis Crick received Nobel prize for describing the structure of DNA in Physiology of Medicine.Basic principles to extract DNA from cells are:
Clean raw cells
Break the cells (lysis) to release DNA into solution
Destroy membrane, proteins and lipids that may bind and destroy the DNA
precipitate DNA by using alcohol
Lets extract DNA from onion.
Materials:
blender, salt, shampoo or soap, tea or coffee filter, ethanol of isopropanol, warm water, tooth pick.
Methods:
clean the onion. Cut into pieces, put into blender, add salt and water. Blend for 3-5 seconds. Filter it using tea or coffee filter. Add 2 or 3 spoon of shampoo. Stir gently so that there will be no bubble. Slowly pour ice cold alcohol into the glass. So there will be two layer. Wait for 3-5 minutes. You will see some white DNA at the surface of the top layer. You can collect this layer by tooth pics and put into another tube with fresh water. Within one minute the DNA will be invisible again in the new tube with water.
If you have questions and if you disagree with anything I have written here just mail me.
See also,
http://library.thinkquest.org/19037/dna_extraction.html
http://en.wikipedia.org/wiki/User:Seans_Potato_Business/DNA
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