10K Genes/Dirt Cheap Miniprep

Introduction

In order to process hundreds of different plasmids, we needed an alternative to traditional miniprep columns that was automatable and affordable. Normally, miniprep procedures use silica membranes in order to bind the DNA after an alkali lysis step. Silica membranes are basically single use, due to some DNA being leftover in the membrane after the final wash. Though efficient, this means that they are often very expensive (~$1 per column).

In 2011, Dr. Lezin characterized a one-step miniprep procedure that uses Non-ionic detergents (NIDs) and simple enzymes , which makes this method approximately 2 orders of magnitude cheaper than the silica membrane methods (~¢1 per reaction if reagents are sourced from Sigma).

However, since this method uses commercially available chemicals and enzymes, this method can further be reduced in price to the point of being 2 orders of magnitude lower than the price than if the reagents were purchased from Sigma (~¢.01 per reaction if reagents are sourced from Amazon and Alibaba).

Overview

This is the Dirt Cheap (DC) Miniprep protocol, an alternate miniprep protocol that has the potential to be much cheaper and easier to automate than traditional silica based purification protocols. It uses commercially available chemicals and enzymes in order to lower the price of each miniprep by at least an order of magnitude.

Materials

DC Miniprep Buffer

DC Buffer composition

• 0.75M NH₄Cl
• 5mM EDTA
• 0.15% Triton X-100

DC Buffer recipe per 500mL

• 20.05g NH₄Cl
• 0.7306g EDTA
• 750ul Triton X-100
• 500mL H₂O

DC Miniprep Enzyme Mix

1x Final DC enzyme concentration

• 100ug/ml lysozyme
• 25ug/ml RNAseA

100x DC Enzyme recipe per 1mL

• 10mg lysozyme
• 2.5mg RNAseA
• 1mL H₂O

Procedure

Koeng (talk) 15:15, 10 January 2018 (PST) Until test and verify our own automated methods, the below protocol is shamelessly ripped from "A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles".

Long Protocol

1. 1.5-2 ml of bacterial cultures were pelleted at 6000-7000 rpm for 1 min.
2. After drawing 150 µl extraction buffer into a pipette tip, the pellet was loosened off the tube wall with the tip without releasing the buffer. Then the extraction buffer was added and the pellet resuspended.
3. The bacterial suspension was incubated at 65°C for 5 min.
4. Suspensions were centrifuged at maximum rpm for 10 min or until a tight bacterial pellet was formed. The pellet was removed with a toothpick.
5. 100-120 µl isopropanol was added, followed by mixing and centrifugation of the solution at 7000 rpm for 10 min at RT.
6. DNA usually forms film-like precipitates that adhere well to tube walls and are invisible in isopropanol solutions. After discarding the supernatant, the DNA was centrifuged after adding 70% ethanol. Ethanol was removed, and the DNA pellet was dissolved in 20-50 µl TE buffer.

Short (Crude) Protocol

1. 1.5-2 ml of bacterial cultures were pelleted at 6000-7000 rpm for 1 min.
2. After drawing 150 µl extraction buffer into a pipette tip, the pellet was loosened off the tube wall with the tip without releasing the buffer. Then the extraction buffer was added and the pellet resuspended.
3. The bacterial suspension was incubated at 65°C for 5 min.
4. Suspensions were centrifuged at maximum rpm for 10 min or until a tight bacterial pellet was formed. The supernatant was used as crude DNA preps.

Notes

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

1. Koeng (talk) 15:08, 10 January 2018 (PST) Make sure to autoclave the DC Buffer before use.

Contact

• Keoni Gandall - koeng101@gmail.com