Luckau Protocols:Low TE: Difference between revisions
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[ | {|{{table}} width="900" | ||
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| width="600" style="background-color: #BCED91;" align="center"| <span style="font-size:32px;"> Low TE Protocol </span> | |||
|align="left" style="background-color: #9DB68C;" | | |||
<span style="font-size:16px;"> [[User:Tara_K._Luckau | Tara K. Luckau's Home Page]] </span> | |||
[[User:Tara_K._Luckau/Notebook/Team_ConGen | Conservation Genetics Lab Notebook]] | |||
[[Luckau_Protocols | Tara's Protocols]] | |||
|} | |||
==Purpose== | ==Purpose== | ||
Low TE buffer is used to store DNA. | Low TE buffer is used to store DNA. | ||
* EDTA chelates Mg<sup>2+</sup> and other divalent metals ions (inhibits DNAse and RNAse | * EDTA chelates Mg<sup>2+</sup> and other divalent metals ions | ||
**(inhibits DNAse and RNAse to suppress DNA and RNA degradation) | |||
* Tris is a buffering agent to keep the solution at a defined pH | * Tris is a buffering agent to keep the solution at a defined pH | ||
** The 'Cl' or 'HCl' indicates the pH was attained using aqueous hydrochloric acid (HCl), since Tris is naturally basic | |||
== | ==Protocol== | ||
1x Low TE | 1x Low TE = 10 mM Tris-HCl + 0.1 mM EDTA | ||
{| {{table}} | |||
|+ ''' | {| {{table}} style="text-align:center" | ||
|+ '''to make 1x Low TE:''' | |||
|- | |- | ||
! Reagent !! [Final] !! to make 1 L !! to make 500 mL | |||
|- | |- | ||
| | |0.5M Tris-HCl, pH 8.0 || 10 mM || 20 mL || 10 mL | ||
|- | |- | ||
|0.5M EDTA, pH 8.0 || 0.1 mM || 200 µL || 100 µL | |||
|- | |- | ||
|ddH<sub>2</sub>O || || 979.8 mL || 489.9 mL | |||
|} | |} | ||
*Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle | |||
*Store at 4°C or room temperature | |||
==FYI== | ==FYI== | ||
* Note, downstream reactions (PCR) typically require Mg<sup>2+</sup>, potentially making the presence of EDTA in the reaction problematic. So, when using DNA or RNA that was suspended in TE, keep track of the amount of EDTA in the mix to make sure there is still enough Mg<sup>2+</sup> for subsequent reactions to proceed successfully. Each EDTA molecule chelates one Mg<sup>2+</sup> ion. | * Note, downstream reactions (PCR) typically require Mg<sup>2+</sup>, potentially making the presence of EDTA in the reaction problematic. So, when using DNA or RNA that was suspended in TE, keep track of the amount of EDTA in the mix to make sure there is still enough Mg<sup>2+</sup> for subsequent reactions to proceed successfully. Each EDTA molecule chelates one Mg<sup>2+</sup> ion. | ||
* | *TE versus Low TE: | ||
::{| {{table}} style="text-align:center" | |||
| - || Tris-Cl || EDTA | |||
|- | |||
| TE || 10 mM || 1 mM | |||
|- | |||
|Low TE || 10 mM || 0.1 mM | |||
|} | |||
*Some people use TE buffers with different pH's for different applications | *Some people use TE buffers with different pH's for different applications | ||
**DNA is stored at pH 8 to reduce depurination, which is acid catalyzed | **DNA is stored at pH 8 to reduce depurination, which is acid catalyzed | ||
**RNA is stored at | **RNA is stored at pH 7.5 to reduce degradation of RNA, which is base-catalyzed | ||
**Most downstream reactions will not be influenced by the slightly different pH storage conditions. | **Most downstream reactions will not be influenced by the slightly different pH storage conditions. | ||
==Sources== | |||
*OpenWetWare's Materials [[TE]] page | |||
*[http://cshprotocols.cshlp.org/cgi/content/full/2006/3/pdb.rec8018 Cold Spring Harb Protoc; 2006; doi:10.1101/pdb.rec8018] | |||
*[http://www.cytographica.com/lab/solutions/TE.htm Cytographica] | |||
Latest revision as of 16:02, 28 October 2011
| Low TE Protocol |
Purpose
Low TE buffer is used to store DNA.
- EDTA chelates Mg2+ and other divalent metals ions
- (inhibits DNAse and RNAse to suppress DNA and RNA degradation)
- Tris is a buffering agent to keep the solution at a defined pH
- The 'Cl' or 'HCl' indicates the pH was attained using aqueous hydrochloric acid (HCl), since Tris is naturally basic
Protocol
1x Low TE = 10 mM Tris-HCl + 0.1 mM EDTA
| Reagent | [Final] | to make 1 L | to make 500 mL |
|---|---|---|---|
| 0.5M Tris-HCl, pH 8.0 | 10 mM | 20 mL | 10 mL |
| 0.5M EDTA, pH 8.0 | 0.1 mM | 200 µL | 100 µL |
| ddH2O | 979.8 mL | 489.9 mL |
- Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle
- Store at 4°C or room temperature
FYI
- Note, downstream reactions (PCR) typically require Mg2+, potentially making the presence of EDTA in the reaction problematic. So, when using DNA or RNA that was suspended in TE, keep track of the amount of EDTA in the mix to make sure there is still enough Mg2+ for subsequent reactions to proceed successfully. Each EDTA molecule chelates one Mg2+ ion.
- TE versus Low TE:
- Tris-Cl EDTA TE 10 mM 1 mM Low TE 10 mM 0.1 mM
- Some people use TE buffers with different pH's for different applications
- DNA is stored at pH 8 to reduce depurination, which is acid catalyzed
- RNA is stored at pH 7.5 to reduce degradation of RNA, which is base-catalyzed
- Most downstream reactions will not be influenced by the slightly different pH storage conditions.
Sources
- OpenWetWare's Materials TE page
- Cold Spring Harb Protoc; 2006; doi:10.1101/pdb.rec8018
- Cytographica
