Luckau Protocols:Low TE: Difference between revisions

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(New page: ==Purpose== TE buffer is often used to store DNA and RNA. * EDTA in TE chelates Mg<sup>2+</sup> and other divalent metals ions necessary for most causes of DNA and RNA degradation, su...)
 
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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==
TE buffer is often used to store DNA and RNA.  
Low TE buffer is used to store DNA.  
* [[EDTA]] in TE chelates Mg<sup>2+</sup> and other divalent metals ions necessary for most causes of DNA and RNA degradation, suppressing these processes.
* EDTA chelates Mg<sup>2+</sup> and other divalent metals ions
* [[Tris]] is a buffering agent to keep the solution at a defined pH.
**(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


==Recipe 10x TE ==


{| {{table}}
==Protocol==
|+ '''for 1 liter of 10x TE stock solution'''
1x Low TE = 10 mM Tris-HCl + 0.1 mM EDTA
 
 
{| {{table}} style="text-align:center"
|+ '''to make 1x Low TE:'''
|-
|-
|volume || reagent || final conc.
! Reagent !! [Final] !! to make 1 L !! to make 500 mL
|-
|-
|100 ml || 1M Tris-HCl pH 7.5 or 8.0 (see notes) || 100 mM
|0.5M Tris-HCl, pH 8.0 || 10 mM || 20 mL || 10 mL
|-
|-
|20 ml  || 0.5M EDTA pH 8.0 || 10 mM
|0.5M EDTA, pH 8.0 || 0.1 mM || 200 µL || 100 µL
|-
|-
|880 ml || ddH<sub>2</sub>O ||
|ddH<sub>2</sub>O ||   || 979.8 mL || 489.9 mL
|}
|}


==Recipe 1x TE ==


{| {{table}}
*Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle
|+ '''for 1 liter of 1x TE solution'''
*Store at 4°C or room temperature
 
 
==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.
*TE versus Low TE:
::{| {{table}} style="text-align:center"
| - || Tris-Cl || EDTA
|-
|-
|volume || reagent || final conc.
| TE || 10 mM || 1 mM
|-
|-
|10 ml || 1M Tris-HCl pH 7.5 or 8.0 (see notes) || 10 mM
|Low TE || 10 mM || 0.1 mM
|-
|2 ml  || 0.5M EDTA pH 8.0 || 1 mM
|-
|988 ml || ddH<sub>2</sub>O ||
|}
|}
*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.


→ 1x TE is 10 mM [[Tris]]-HCl and 1 mM [[EDTA]]
==Notes==
*For the Tris-HCl use Tris base and adjust to desired pH using HCl.
*TE buffer is often used to store DNA and RNA. The [[EDTA]] in TE chelates Mg<sup>2+</sup> and other divalent metals ions necessary for most causes of DNA and RNA degradation, suppressing these processes. However, downstream reactions like restriction digests, PCR, ligations, and reverse transcription 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, you should 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.
*Some protocols use TE 10:0.1 with 0.1 mM EDTA to reduce the interaction of the EDTA with downstream applications.
*Some people use TE buffers with different pH's for different applications. For example, DNA is stored at pH 8 to reduce depurination, which is acid catalyzed, while RNA is stored at a slightly lower pH (7.5) because degradation of RNA is base-catalyzed. Most downstream reactions will not be influenced by the slightly different pH storage conditions.
*For dilution of primers water for injections can be used rather than TE.


==See also==
==Sources==
* [http://cshprotocols.cshlp.org/cgi/content/full/2006/3/pdb.rec8018 10x TE buffer at Cold Spring Harbor Protocols]
*OpenWetWare's Materials [[TE]] page
* [http://www.cytographica.com/lab/solutions/TE.htm 10x and 1x TE at cytographica]
*[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

Tara K. Luckau's Home Page

Conservation Genetics Lab Notebook

Tara's Protocols


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


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
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

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