Jan 30, 2025

Public workspacePlasmid Cloning - PureLink Kit

DOI
dx.doi.org/10.17504/protocols.io.q26g7m988gwz/v1
  • 1University of Minnesota
Ty Flanagan
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DOI: dx.doi.org/10.17504/protocols.io.q26g7m988gwz/v1
Protocol Citation: Ty Flanagan 2025. Plasmid Cloning - PureLink Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7m988gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 29, 2025
Last Modified: January 30, 2025
Protocol Integer ID: 119307
Abstract
This protocol is for plasmid cloning using E. coli and the PureLink Quick Plasmid Miniprep Kit. See the Plasmid Ext - PureLink Quick Plasmid Miniprep Kit Protocol.pdf on the Goolge Drive.
Grow modified E. coli overnight
Grow modified E. coli overnight
12m
12m
Prepare working space for bacterial work.
  • 70% ethanol spray down.
  • Bunsen burner.
  • Gloves.
  • Follow aseptic technique.
5m
Prepare 2 tubes withAmount2.5 mL LB liquid broth with 50 ug/mL of ampicillin.
Note
Use a non-sealing tube, or at the very end, before incubation, keep the tube cap slightly unscrewed.

The 5 mL non-sealing tubes are recommended.



3m
Inocculate the media with one "tip-worth" of frozen bacterial stock. Use a 1000 uL pipette tip to physically scrape the frozen stock to collect the bacteria in the pipette tip's tip. Mix the media with the pipette tip.
2m
Incubate the culture at Temperature37 °C in the shaking incubator overnight.
Note
Keep volumes low relative to tube size as to prevent spilling. Also, make sure that the tube is supported as to not move much.
Do not overtighten screw-on caps.

2m
Incubation
Overnight
Follow PureLink Quick Plasmid Miniprep Kit Protocol
Follow PureLink Quick Plasmid Miniprep Kit Protocol
58m
58m
Begin heating Amount200 µL of TE Buffer in a 1.5 mL tube at Temperature70 °C using a dry bath.

5m
Centrifuge the cultures and decant the media.
Centrifigation12000 x g, 00:03:00
5m
Add Amount250 µL of Resuspension Buffer (R3) and completely resuspend the bacteria pellet using the pipette.
Note
If using a newer kit, make sure that RNase A has been added to the R3 solution.


5m
Add Amount250 µL of Lysis Buffer (L7). Gently mix by inversion 3 times. Incubate at room temp for Duration00:05:00 .

Note
Do not vortex.

7m
Add 350 uL of Precipitation Buffer (N4). Quickly after addition, mix by inversion or vigorous shaking.
Note
Do not vortex.

4m
Centrifuge.
Centrifigation15000 x g, 00:10:00

10m
Transfer the supernatant into a spin column with a 2 mL wash tube. Centrifuge and discard the flowthrough. Reuse the same wash tube until noted.
Centrifigation12000 x g, 00:01:00

5m
Wash the spin column with Amount500 µL of Wash Buffer (W10). Incubate at room temp for Duration00:01:00 . Centrifuge and discard flowthrough.
Centrifigation12000 x g, 00:01:00
Note
If using a newer kit, make sure that ethanol has been added to the W10 solution.

6m
Add Amount700 µL of Wash Buffer (W9). Centrifuge and discard flowthrough.
Centrifigation12000 x g, 00:01:00
Centrifuge and place spin column into new 1.5 mL tube.
Centrifigation12000 x g, 00:01:00
Note
If using a newer kit, make sure that ethanol has been added to the W9 solution.



6m
Add Amount75 µL of the preheated TE Buffer (TE). Incubate at room temp for Duration00:01:00 .
2m
Centrifuge and discard the column.
Centrifigation12000 x g, 00:02:00
Note
Store DNA at Temperature4 °C for short term and Temperature-20 °C for long term. Freeze-thaw cycles damage DNA products, but frozen is better for long term storage. ~4 weeks or 30 days is general guidance on shelf life or refrigerator DNA.


3m