Size selective DNA precipitation

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===Reagents===
===Reagents===
*DNA to be separated
*DNA to be separated
-
*30% (w/v) PEG 8000/30 mM MgCl<sub>2</sub> (concentration of PEG 8000 can be varied to shift the size of the percipitated DNA)
+
*30% (w/v) PEG 8000/30 mM MgCl<sub>2</sub> (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)
*TE Buffer, pH 8.0 (10 mM TRIS-HCl, 1 mM EDTA, pH 8.0)

Revision as of 18:49, 27 August 2008

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 and MgCl2 concentration the range of precipitated DNA fragments can be adjusted.

Materials

Reagents

  • DNA to be separated
  • 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

Troubleshooting

Notes

This protocol is being mention in the manual for the Gateway recombinational cloning system based on published methods [1, 2, 3]

Acknowledgments

Acnkowledge any help you had in development, testing, writing this protocol.

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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