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Miraprep Objective
Author(s):
Pattarin Blanchard
Adapted from Pronobis et al., 2016 supplement 1, this is a plasmid purification protocl that uses miniprep kits and columns and delivers midi/maxi-level DNA yields using a fraction of the reagents and time. Some sources say the name is short for Miracle Prep, but the first author’s first name is also Mira.
While the protocol describes using a large culture with miniprep columns, the key trick is the 1:1 ethanol addition to the lysate supernatant, which increases the DNA carrying capacity of the silica columns. Users report the ethanol addition getting them more than double the yield from a typical miniprep culture volume,.
The standard protocol is for 50 mL culture. See the toggle for a 10 mL version.
Materials
1x 50 mL centrifuge tube
1x 15 mL centrifuge tube
Resuspension buffer, no RNase added
RNase A, 10 mg/mL stock
Miniprep kit components; lysis, neutralization, wash buffers
96% ethanol, molecular biology grade
x1 2 mL microcentrifuge tube
x9 1.5 mL microcentrifuge tubes
x5 spin columns
Ultrapure water
Equipment
Procedure
Pellet a 50 mL overnight (12-16h) culture in a 50 mL centrifuge tube at 4000 g rcf at 4C for 10 minutes.
While waiting, prepare 2 mL resuspension buffer with RNase A to a working concentration of 50 ug/mL in a 2 mL tube.
The Thermo Scientific RNase A stock in the -20 freezer is 10 mg/mL; use 10 uL of this stock
Discard supernatant and in the same tube, add prepared 2 mL resuspension buffer+RNase A to resuspend by vortexing.
Add 2 mL lysis buffer, invert 4 times and incubate for 3 minutes at room temperature.
Add 2 mL of neutralization buffer and invert 3-4 times.
Distribute the lysate into four 1.5 mL microcentrifuge tubes by pouring. Do not pipette to not disturb the lysate and debris.
Pellet debris at 13,200xg rcf for 10 minutes.
Collect supernatants into a 15 mL centrifuge tube and add a 1X volume of ethanol. Mix thoroughly by vortexing for 5 seconds.
Load the mix into 5 spin columns, 700 uL at a time. Spin for 30s at 13,200xg rcf and discard flow-through.
Repeat step 9 until all of the sample has been spun through.
Wash columns by adding 500 uL wash solution per column, spin at 13,200xg rcf for 30 seconds, and discard flow-through. Do this twice .
Dry the columns by spinning at 13,200xg for 1 minute 30 seconds.
Place the columns into 1.5 mL microcentrifuge tubes. For each column, add 30 uL ultrapure water to the centre, and wait 2 minutes.
Spin the columns at 13,200xg rcf for 2 minutes to elute DNA.
Combine the elutes into one tube for a final volume of 150-175 uL.
Measure DNA concentration, see NanoDrop Spectrophotometer .
10 mL version
Materials
2x 15 mL centrifuge tubes
Resuspension buffer, no RNase added
RNase A, 10 mg/mL stock
Miniprep kit components; lysis, neutralization, wash buffers
96% ethanol, molecular biology grade
x3 1.5 mL microcentrifuge tubes
x2 spin columns
Ultrapure water
Equipment
Procedure
Pellet a 10 mL overnight (12-16h) culture in a 15 mL centrifuge tube at 4000xg rcf at 4C for 10 minutes.
While waiting, prepare 400 uL resuspension buffer with 2 uL RNase A for a working concentration of 50 ug/mL in a 1.5 mL tube.
Discard supernatant and in the same tube, add prepared 400 uL resuspension buffer+RNase A to resuspend by vortexing.
Add 400 uL lysis buffer, invert 4 times and incubate for 3 minutes at room temperature.
Add 400 uL of neutralization buffer and invert 3-4 times.
Pellet debris at 13,200xg rcf for 10 minutes. Transfer supernatant to a new 15 mL centrifuge tube.
Add a 1X volume of ethanol and mix thoroughly by vortexing for 5 seconds.
Load the mix into 2 spin columns, 600 uL at a time. Spin for 30s at 13,200xg rcf and discard flow-through.
Repeat step 8 until all of the mix has been spun through.
Wash columns by adding 500 uL wash solution per column, spin at 13,200xg rcf for 30 seconds, and discard flow-through. Do this twice .
Dry the columns by spinning at 13,200xg for 1 minute 30 seconds.
Place the columns into 1.5 mL microcentrifuge tubes. For each column, add 30 uL ultrapure water to the centre, and wait 2 minutes.
Spin the columns at 13,200xg rcf for 2 minutes to elute DNA.
Combine the elutes into one tube for a final volume of ~60 uL.
Measure DNA concentration, see NanoDrop Spectrophotometer .