Protocol Citation: Team Fudan iGEM 2022. Plasmid Miniprep for 2022. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpjwdpgzp/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: October 10, 2022
Last Modified: October 11, 2022
Protocol Integer ID: 71085
Abstract
Plasmid extraction and purification, based on Vazyme Mini-prep Kit manual.
Pellet bacteria culture in a 1.6mL centrifugation tube at 13,000rpm for 1 minute. Decant supernatant. The pellet in the tube can be stored in -20 freezer if time not enough for the following steps.
Add 250uL of resuspension solution (P1 with RNase) and vortex. After adding RNase into the P1, P1 is stored in 4 degree refrigerator.
Add 250uL of lysis solution (P2) and invert gently 4-6 times. P2 contains NaOH, and must keep tightly closed.
Add 350uL of neutralization solution (P3) and invert gently 4-6 times. Do not vortex, which will break bacteria genomic DNA and genomic DNA fragments will contaminate purified plasmids.
Spin at 13,000rpm for 10 minutes, at 4 dgree.
Take the clear supernatant, without any white protein cloud. Bind the DNA to the column by decanting.
Centrifuge at 13,300 rpm for 1 minute.
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Wash the column with 600uL of wash solution (PW2) and centrifuge for 1 minute. Remind adding pure ethanol into PW2 as indicated on the bottle.
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Discard the flow-through, wash again with 600uL of PW2, and centrifuge for 1 minute. Discard the flow-through and centrifuge the column again for 2 minutes.
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3m
Keep the cap open and wait until no ethanol smell. Move the column into a new centrifugation tube. Elute the purified DNA with 50uL of elution buffer (TE; 10 mM Tris-Cl, 1 mM EDTA). Then, centrifuge for 1 minutes. Collect the flow-through, which is the plasmid DNA. Because pET28 based is low-copy plasmid, pre-warm TE to 50 degree helps the elution.
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