Sep 28, 2023
Plasmid Extraction (Plasmid Isolation)
- 1National University of Singapore
Protocol Citation: NUS iGEM 2023. Plasmid Extraction (Plasmid Isolation). protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3yq6l3p/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: September 27, 2023
Last Modified: September 28, 2023
Protocol Integer ID: 88484
Keywords: Plasmid Extraction, Plasmid, Buffer P1, Buffer P2, Buffer N3, Buffer PB, Buffer PE, Plasmid Isolation, Isolation
Abstract
2023 NUS-Singapore iGEM team followed this protocol to isolate the plasmid from the cells.
Protocol materials
Buffer P2QiagenCatalog #19052
Step 7
Buffer PEQiagenCatalog #19065
In 2 steps
Buffer P1QiagenCatalog #19051
Step 5
RNase AQiagenCatalog #19101
Step 5
Buffer N3QiagenCatalog #19064
Step 9
PB bufferQiagenCatalog #19066
Step 15
Safety warnings
- Proper lab PPE must be worn at all times.
Before start
Glycerol stock (900 µL of cell stock in 300 µL of 100% glycerol solution) may be prepared and keep it in the -80 °C fridge before the plasmid extraction procedure.
Cell Culture
Cell Culture
Incubate the cells containing the plasmid of interest in a Falcon tube with 5 mL of LB media and 5 µL of the appropriate antibiotics Overnight at 37 °C before starting the plasmid extraction.
Plasmid Extraction
Plasmid Extraction
5m
5m
Take out the Falcon tube with the cultured cells from the incubator.
Centrifuge the Falcon tube at 5000 rpm, 4°C, 00:05:00 .
5m
Discard the supernatant in the Falcon tube and keep the cell pellet.
Add 250 µL of Buffer P1QiagenCatalog #19051 (with RNase AQiagenCatalog #19101 added, kept in the 4 °C fridge) into the Falcon tube and resuspend the cell pellet.
Transfer the whole solution into a Eppendorf tube.
Add 250 µL of Buffer P2QiagenCatalog #19052 into the Eppendorf tube to lyse the cells.
Shake the Eppendorf tube for mixing.
Add 350μL of Buffer N3QiagenCatalog #19064 into the Eppendorf tube.
Shake the Eppendorf tube for mixing, a cloudy solution shall be observed.
Centrifuge the Eppendorf tube at 13 rpm, 00:10:00 .
10m
Transfer the supernatant into a Mini Prep tube and discard the cell debris.
Centrifuge it at 13 rpm, 00:01:00 .
1m
Discard the flow-through and place the Mini Prep tube back into the same tube.
Add 500 µL of PB bufferQiagenCatalog #19066 into the Mini Prep tube.
Centrifuge it at 13 rpm, 00:01:00 .
Discard the flow-through and place the Mini Prep tube back into the same tube.
Add 700 µL of Buffer PEQiagenCatalog #19065 into the Mini Prep tube.
Centrifuge it at 13 rpm, 00:01:00 .
Discard the flow-through and place the Mini Prep tube back into the same tube.
Centrifuge it at 13 rpm, 00:01:00 to remove the Buffer PEQiagenCatalog #19065 residual.
Transfer the Mini Prep column into the newly labelled Eppendorf tube.
Add 50 µL of DI water into the Eppendorf tube.
Centrifuge it at 13 rpm, 00:01:00 , ensuring that the direction of the Eppendorf tube’s cap is the same as the direction of spinning to avoid breaking.
Discard the Mini Prep column, the solution left in the Eppendorf tube is the plasmid.
Use NanoDrop
Equipment
NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer
NAME
UV-Vis Spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-ONE-W
SKU
to measure the concentration (under "dsDNA" mode) and the purity of the plasmid.
Store the isolated plasmid at room temperature.