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:
- Mix 400µl Sera-mag stock and 600µl ddH2O
- Collect beads on magnet.
- Remove supernatant
- Resuspend beads in 1ml 0.5M EDTA pH 7.2.
- Place tube back on magnet. Remove supe.
- Resuspend beads in 1ml 0.5M EDTA pH 7.2 again.
Store at 4˚C.
Purifying DNA:
- Spin down 1 ml bacterial culture.
- Resuspend pellet in 30µl Solution 1.
- 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.
- Neutralize with 45µl Solution 3; mix by inversion.
- Spin
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- Anecdotal observations that might be of use to others can also be posted here.
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References
Relevant papers and books
- 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
- Who has experience with this protocol?
or instead, discuss this protocol.