Lane:MagneticPlasmidPrep

Overview

This is an (as yet untested) work-in-progress protocol with the goal of replacing silica columns for bacterial minipreps with a magnetic bead based protocol. The starting point is ref. [1]; the protocol is based on this. It may be possible to simplify the cleared lysate generation to a protocol more similar to that of Zymo's direct-from-culture lysis.

Materials

• Solution 1 (Use 30µl/prep - make 10ml; store at RT or at 4˚C to preserve RNAse A if preferred):
  • 50mM Glucose
  • 25mM Tris pH 8.0
  • 10mM EDTA pH 8.0
  • 100µg/ml RNAse A

Procedure

Preparing magnetic bead stock Carboxy-coated magnetic beads are Sera-mag SpeedBeads (Fisher #09-981-123). These come at 50mg/ml; protocol calls for 20mg/ml in 0.5M EDTA pH7.2:

1. Mix 400µl Sera-mag stock and 600µl ddH₂O
2. Collect beads on magnet.
3. Remove supernatant
4. Resuspend beads in 1ml 0.5M EDTA pH 7.2.
5. Place tube back on magnet. Remove supe.
6. Resuspend beads in 1ml 0.5M EDTA pH 7.2 again.

Store at 4˚C.

Purifying DNA:

1. Spin down 1 ml bacterial culture.
2. Resuspend pellet in 30µl Solution 1.
3. Add 60µl Solution 2. Mix by inversion, make snot, incubate for a minute or two. More than five minutes is reported to permanently damage DNA.
4. Neutralize with 45µl Solution 3; mix by inversion.
5. Spin

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

1. Anecdotal observations that might be of use to others can also be posted here.

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References

Relevant papers and books

1. Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. DOI:10.1073/pnas.78.11.6840 |

Contact

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