Sebastian Cocioba
posted 3 months ago
Amateur šŸŒæ Biologist ā€¢ Aspiring šŸŒ» Designer Independent Researcher at Binomica Labs Degreeless Heathen #diybio ā€¢ #homelab šŸ§¬šŸ“–: ATinyGreenCell.com šŸ¦£:@ATinyGreenCell@genomic.social šŸ¦:@ATinyGreenCell
Method

Columnless Old-School Miniprep

This is my go-to alakline lysis miniprep protocol that does not require a kit or silica columns. It's based on the tried and true protocols of yesteryear and brought to my attention a few years ago by J. Leon (@jesussaur) on Twitter. I've been using it ever since and have successfully sent plasmids out for sequencing via plasmidsaurus (nanopore whole plasmid sequencing) with good results. The prep is by no means utterly devoid of any other DNA or protein contaminants but it is good enough for routine lab work including goldengate type2S cloning. One pro tip is to orient your tubes in the same direction every time you spin them so the pellet forms in the same place. I like keeping the hinges of the 1.5mL tubes facing outward from the center of the centrifuge to visually confirm the same orientation. This step is especially important during the post-alcohol spindowns and final removal of residual alcohol.

Columnless Miniprep Buffers

Resuspension Buffer Tris-HCL 50mM EDTA 10mM RNaseA 100ug/mL (IMPORTANT) pH 8.0 Store at 4C

Lysis Buffer NaOH 200mM SDS 1% Store air-tight at RT

Neutralization Buffer Potassium Acetate 3M pH 5.5 adjusted with Glacial Acetic Acid Store air-tight at RT

Ethanol 70%

Isopropanol 100%

Columnless Miniprep Protocol

Description: A simple method for isolating plasmid DNA from bacterial cultures without silica columns or harsh reagents.

  1. Grow a single colony of your plasmid carrying E. coli strain in a small volume of LB + appropriate antibiotics overnight shaken at 37C.

  2. The next day, spin down 1mL of bacterial culture in a 1.5mL tube at maximum speed for 1 minute.

  3. Remove the supernatant carefully and respend the cells in 100uL of Resuspension Buffer.

  4. Add 100uL of Lysis Buffer. Invert 10x. Incubate at room temp for 2 mins.

  5. Add 120uL of Neutralization Buffer. Invert 10x. Incubate at room tmep for 2 mins.

  6. Spind down tube at maximum speed (~10kG or 14krpm) for 10 minutes.

  7. Carefully decant the supernatant into a new tube. Avoid picking up any cellular debris. Go slow and use smaller pipette tips if need be.

  8. Add 200uL of Isopropanol 100%. Vortex for 10 seconds. Spin down at maximum speed for 10 mins.

  9. Decant the isoporpanol and add 500uL of Ethanol 70%. Vortex. Spin max speed 5 mins.

  10. Decant the enthanol carefully and spin down tube again at max speed for 3 minutes to pull residual ethanol from pellet.

  11. Remove any residual ethanol with a 10uL pipette tip until no liquid remains visible. Air dry the tube at room temp for 10 minutes.

  12. Resuspend plasmid DNA in sterile DH2O and measure concentration and/or run a sample on a gel to determine quality of isolation.

5
Nick Gervais3 months ago

Wicked! Is there some reason you use this over a kit? Cost?

Sebastian Cocioba3 months ago

@Nick Gervais, mostly cost. It works fine for routine plasmid preps, just know that if you're using NEB Turbos to NOT let it grow more than 12hrs or they will start eating your plasmid in late stationary phase. I even do routine Golden Gate assembly using columnless preps as donor plasmids with great success. Sequencing is a bit dirty but if you get high yield the amount of reads via services like Plasmidsarus (nanopore) still yield great seq results. Mostly just really small ecoli frags from chromosome as carryover debris.

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