DNA Precipitation: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
|||
| (18 intermediate revisions by 7 users not shown) | |||
| Line 1: | Line 1: | ||
{{back to protocols}} | |||
==Overview== | ==Overview== | ||
This protocol can be used to concentrate DNA, or to change the buffer the DNA is suspended in. It can also be coupled with phenol chloroform extraction for the purifying nucleic acids. This protocol also works for RNA precipitation (take care to use RNAse free materials in this case). | |||
==Materials== | ==Materials== | ||
*3M NaOAc pH 5.2 | *3M NaOAc pH 5.2 | ||
| Line 11: | Line 12: | ||
==Procedure== | ==Procedure== | ||
#1 | # Add 0.1 volumes of 3M Sodium Acetate solution to 1 volume of DNA sample. | ||
#2 volumes EtOH | # Add 1ul Glycogen to the DNA sample. | ||
#-20°C | # Add 2 volumes of 95% EtOH to the DNA Sample. | ||
#Centrifuge | # Store the solution overnight at -20°C or for 30 minutes at -80°C. | ||
# Centrifuge the solution at maximum speed for least 15 minutes. | |||
# Decant and discard the supernatant. | |||
# (Optional) Add 1 ml of 70% EtOH to the pellet and let sit for 5 minutes. | |||
# (Optional) Centrifuge the sample at maximum spped for 5 minutes. | |||
# (Optional) Decant and Discard the supernatant. | |||
# Air-dry the pellet for 10-15 minutes at room temperature until all liquid is gone. | |||
# Resuspend in desired volume of water or buffer | |||
==Notes== | |||
*'''[[User:Ryan R Hurtado|Ryan R Hurtado]] 17:21, 22 April 2008 (EDT)''': | |||
-20°C for an hour is fine for using larger (1mL of bacterial culture, plasmid) amounts of DNA | |||
The DNA pellet will not always be visible depending on how much DNA you are precipitating. So always take care in loading your samples in the centrifuge to remember the direction they are facing. The DNA pellet will be on the part of the tube facing the outside of the centrifuge. | |||
This protocol will precipitate all nucleic acids, not just DNA. If you do not want RNA in your sample, one of the many ways to deal with it is to simply resuspend in TE + RNAse at the last step and leave it at room temperature for 15mins-1hr. | |||
==BioCoder version== | |||
Following is the DNA Precipitation protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth|Vaishnavi]] | |||
====Text Output==== | |||
[[DNA Precipitation protocol]] | |||
====Source Code==== | |||
[[DNA Precipitation protocol - source code]] | |||
==References== | ==References== | ||
==Links== | |||
*[http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/ basics and discussion] | |||
==Contact== | ==Contact== | ||
[[Talk:{{PAGENAME}}|Discuss this protocol]]. | |||
<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> | <!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> | ||
[[Category:Protocol]] | [[Category:Protocol]] | ||
[[Category:DNA]] | |||
[[Category:In vitro]] | |||
[[Category:Needs attention]] <!--Delete this line once the protocol is complete--> | [[Category:Needs attention]] <!--Delete this line once the protocol is complete--> | ||
Latest revision as of 02:43, 19 November 2009
| back to protocols | ||
Overview
This protocol can be used to concentrate DNA, or to change the buffer the DNA is suspended in. It can also be coupled with phenol chloroform extraction for the purifying nucleic acids. This protocol also works for RNA precipitation (take care to use RNAse free materials in this case).
Materials
- 3M NaOAc pH 5.2
- EtOH 95%
- Glycogen (optional)
Procedure
- Add 0.1 volumes of 3M Sodium Acetate solution to 1 volume of DNA sample.
- Add 1ul Glycogen to the DNA sample.
- Add 2 volumes of 95% EtOH to the DNA Sample.
- Store the solution overnight at -20°C or for 30 minutes at -80°C.
- Centrifuge the solution at maximum speed for least 15 minutes.
- Decant and discard the supernatant.
- (Optional) Add 1 ml of 70% EtOH to the pellet and let sit for 5 minutes.
- (Optional) Centrifuge the sample at maximum spped for 5 minutes.
- (Optional) Decant and Discard the supernatant.
- Air-dry the pellet for 10-15 minutes at room temperature until all liquid is gone.
- Resuspend in desired volume of water or buffer
Notes
- Ryan R Hurtado 17:21, 22 April 2008 (EDT):
-20°C for an hour is fine for using larger (1mL of bacterial culture, plasmid) amounts of DNA
The DNA pellet will not always be visible depending on how much DNA you are precipitating. So always take care in loading your samples in the centrifuge to remember the direction they are facing. The DNA pellet will be on the part of the tube facing the outside of the centrifuge.
This protocol will precipitate all nucleic acids, not just DNA. If you do not want RNA in your sample, one of the many ways to deal with it is to simply resuspend in TE + RNAse at the last step and leave it at room temperature for 15mins-1hr.
BioCoder version
Following is the DNA Precipitation protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi
Text Output
Source Code
DNA Precipitation protocol - source code
