Jun 07, 2023
Version 2
TIANprep Mini Plasmid Kit Protocol V.2
- TIANGEN Biotech1
- 1TIANGEN Biotech (Beijing) Co., Ltd.
Protocol Citation: TIANGEN Biotech 2023. TIANprep Mini Plasmid Kit Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpj4n5gzp/v2Version created by Chottiwatt Jittprasong
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
In Use
Created: June 06, 2023
Last Modified: June 07, 2023
Protocol Integer ID: 82947
Keywords: plasmid DNA, purification of plasmid dna, tianprep mini plasmid kit, plasmid dna, high quality plasmid dna, adsorption of dna, purification, dna, silica membrane, including restriction enzyme digestion
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Abstract
TIANprep Mini Plasmid Kit is based on alkaline lysis technology followed by adsorption of DNA onto silica membrane in the presence of high salt. Plasmid DNA purified with TIANprep Mini Plasmid Kit is immediately readyfor use. Phenol extraction and ethanol precipitation are not required. High quality plasmid DNA is eluted in a small volume of Tris Buffer or deionized water. This protocol is designed for purification of plasmid DNA from 1-5ml overnight cultures of E. coli in LB (Luria-Bertani) medium. Plasmid DNA prepared by TIANprep Mini Plasmid Kit is suitable for avariety of routine applications including restriction enzyme digestion, sequencing, library screening, ligation and trans-formation, in vitro translation, and transfection of robust cells.
Guidelines
Applicable to TIANprep Mini Plasmid Kit manufactured by TIANGEN.
Materials
RNase A, Buffer BL, Buffer P1, Buffer P2, Buffer P3, Buffer PD, Buffer PW, Buffer EB, Spin Columns CP3, Collection Tubes 2ml
Troubleshooting
Safety warnings
Avoid direct contact with Buffer P2 and P3.
Before start
Buffer P1 must be activated with RNase A solution before use (one vial per one bottle) and store at 2-8 degrees celsius after activation.
Preparation of Bacterial Cells
16m
Centrifiguation of 1-5 mL of bacterial cells in a microcentrifuge tube at 12000 rpm, Room temperature, 00:01:00 , table-top microcentrifuge
Note
Amount of bacterial cells to be added can be divided into two parts rather than being added at once. For example, 4 mL of bacterial cells can be divided to 2 mL and centrifuged before discarding the flow-through discarded and add another 2 mL and centrifuged again to maximize the yield.
1m
Direct drainage of supernatant by opening and inverting the tube.
Expected result
Bacteria pellet should be present in the microcentrifuge tube.
Complete resuspension of the bacteria pellet in 250 µL of Buffer P1.
Note
- Buffer P1 must be pre-activated with RNase A.
- No cell clumps should be visible after resuspension. Vortex and pipette can be used to help homogenize the mixture.
Add 250 µL of Buffer P2 (lysis buffer) and mix by inverting the tube 6-8 times.
Safety information
Avoid direct contact with Buffer P2
Note
- DO NOT vortex or violently mix the reaction.
- DO NOT allow the lysis reaction to continue for more than
5m
Add 350 µL of Buffer P3 and mix immediately by inverting the tube 6-8 times to neutralize Buffer P2.
Safety information
Avoid direct contact with Buffer P3
Expected result
There should be no white precipitation left in the supernatant. If there is, the mixture should be centrifuged again.
Centrifuge at 12000 rpm, Room temperature, 00:10:00 , table-top microcentrifuge
10m
Preparation of Spin Column CP3
1m
Place Spin Column CP3 in a clean collection tube.
Add 500 µL of Buffer BL to CP3.
Centrifuge at 12000 rpm, Room temperature, 00:01:00 , table-top microcentrifuge
1m
Discard the flow-through and put the Spin Column CP3 back into the collection tube.
Plasmid Extraction
9m
Transfer of supernatant from Step 6 to Spin Column CP3 with collection tube attached.
Centrifuge at 12000 rpm, Room temperature, 00:01:00 , table-top microcentrifuge
1m
Discard the flow-through, retain Spin Column CP3 with collection tube attached.
OPTIONAL: Wash the Spin Column CP3 by adding 500 µL of Buffer PD and centrifuge at 12000 rpm, Room temperature, 00:01:00 , table-top microcentrifuge and discard the flow-through.
Note
Recommended to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content.
1m
Wash the column with Buffer PW
Add 600 µL of Buffer PW
Note
Buffer PW must be pre-treated with 96-100% ethanol.
Centrifuge at 12000 rpm, Room temperature, 00:01:00 , table-top microcentrifuge .=
1m
Discard the flow-through, retain Spin Column CP3 with collection tube attached.
Repeat Step 15.
Centrifuge at 12000 rpm, Room temperature, 00:02:00 , table-top microcentrifuge to remove residual Buffer PW.
2m
Air dry the column at room temperature for a while to allow residual ethanol from Buffer PW to evaporate.
Note
Residual ethanol from Buffer PW may inhibit subsequent enzymatic reactions.
Place Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. Discard the collection tube.
Add 50-100 µL of Buffer EB to the center of the Spin Column CP3.
Incubate for 00:02:00
2m
Centrifuge at 12000 rpm, Room temperature, 00:02:00 , table-top microcentrifuge
2m
Protocol references