Size selective DNA precipitation

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==Critical steps==
==Critical steps==
*Before centrifugation mark the tubes in order to know where the pellet will be expected afterwards, as the pellet will be (nearly) invisible
*Before centrifugation mark the tubes in order to know where the pellet will be expected afterwards, as the pellet will be (nearly) invisible
 +
 +
==Other PEG Concentrations and Approximate Size Exclusion==
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*% PEG  Fragments Excluded
 +
*10% --  <300bp
 +
*6%  --  <500bp
 +
*5%  --  <700bp
 +
*4%  --  <1kb
 +
 +
*Note: Yield decreases considerably when <10% PEG is used
 +
==Troubleshooting==
==Troubleshooting==

Revision as of 17:00, 6 January 2012

Contents

Curators

Kersten S. Rabe

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Abstract

A very fast and easy method for the size-selective removal of smaller DNA from larger fragments. By adjusting the PEG concentration the range of precipitated DNA fragments can be adjusted.

Materials

Reagents

  • DNA to be separated (e.g. PCR reaction mixture)
  • 30% (w/v) PEG 8000/30 mM MgCl2 (concentration of PEG 8000 can be varied to shift the size of the percipitated DNA. The concentration used here will remove DNA fragments with less than 300bp)
  • TE Buffer, pH 8.0 (10 mM TRIS-HCl, 1 mM EDTA, pH 8.0)

Equipment

  • Centrifuge which can do up to 10.000 rcf (=g)
  • Appropriate tubes for the centrifuge
  • Pipettes
  • Vortexer

Procedure

  • Mix 50 μL of sample with 150 µL of TE
  • Add 100 µL of PEG/MgCl2
  • Vortex
  • Centrifuge 15 min at 10.000 rcf at roomtemperature
  • Carefully remove supernatant not to disturb the pellet, which will be invisible
  • Dissolve the pellet in a appropriate amount of buffer of choice

Critical steps

  • Before centrifugation mark the tubes in order to know where the pellet will be expected afterwards, as the pellet will be (nearly) invisible

Other PEG Concentrations and Approximate Size Exclusion

  • % PEG Fragments Excluded
  • 10% -- <300bp
  • 6% -- <500bp
  • 5% -- <700bp
  • 4% -- <1kb
  • Note: Yield decreases considerably when <10% PEG is used


Troubleshooting

Notes

  • This protocol is being mentioned in the manual for the Gateway recombinational cloning system based on published methods.[1, 2, 3]
  • Adjusting the PEG concentration can shift the threshold of the size of the precipitated DNA. The higher the final PEG concentration, the smaller the fragments that will be removed (which will stay in the supernantant).
  • If the sample volume is different, simply adjust the other volumes accordingly to end up with the same ratio.

References

  1. http://www.invitrogen.co.jp/focus/181027.pdf [Website]
  2. Paithankar KR and Prasad KS. . pmid:2030954. PubMed HubMed [Paithankar]
  3. Lis JT. . pmid:6246357. PubMed HubMed [Lis]
All Medline abstracts: PubMed HubMed


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BioCoder version

Following is the Size selective 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

Size selective DNA precipitation protocol

Source Code

Size selective DNA precipitation protocol - source code

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