Size selective DNA precipitation
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This protocol is being mention in the manual for the Gateway recombinational cloning system based on published methods <cite>Website Paithankar Lis</cite>
This protocol is being mention in the manual for the Gateway recombinational cloning system based on published methods<cite>Website Paithankar Lis</cite>
Revision as of 18:51, 27 August 2008
Kersten S. Rabe
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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.
- 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)
- Centrifuge which can do up to 10.000 rcf (=g)
- Appropriate tubes for the centrifuge
- Mix 50 μL of sample with 150 µL of TE
- Add 100 µL of PEG/MgCl2
- 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
- Before centrifugation mark the tubes in order to know where the pellet will be expected afterwards, as the pellet will be (nearly) invisible
This protocol is being mention 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.
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