Mar 13, 2023
Fast-n-Easy Plasmid Mini-Prep Kit (cellco)
This protocol is a draft, published without a DOI.
- 1Independent Researcher
Protocol Citation: Guilherme Barbosa 2023. Fast-n-Easy Plasmid Mini-Prep Kit (cellco). protocols.io https://protocols.io/view/fast-n-easy-plasmid-mini-prep-kit-cellco-cq3tvynn
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: March 13, 2023
Last Modified: March 13, 2023
Protocol Integer ID: 78675
Disclaimer
This protocol is from the DATA SHEET of the following kit of cellco:
Fast-n-Easy Plasmid Mini-Prep Kit
Column based isolation of plasmid DNA
Cellco Biotec do Brasil Ltda
Rua Miguel João, 930 | São Carlos - São Paulo |CEP: 13562-180 | Tel.: +55-16-3416-2257
http://www.cellco.com.brPage 1 of 2
Last update: 08/2021
Abstract
Description:
Fast-n-Easy Plasmid Mini-Prep Kit is designed for isolation of
high-purity plasmid or cosmid DNA from bacterial cells for
subsequent amplification, sequencing, restriction digests or
transformations. The 2-step alkaline lysis procedure and binding
column based preparation provide a fast, easy and efficient way
of DNA isolation without shearing or significant loss of product. It
allows elution in a small volume of low-salt buffer. Time-
consumingphenol-chloroformextractionoralcohol
precipitation is not required.
Attachments
DPK_104_en.pdf
501KB
Guidelines
For research use only!
Materials
Kit Contents:
Lysis Buffer including pH indicator
Neutralization Buffer (before use, add RNase A and store at 4°C)
RNase A (store at -20°C)
Activation Buffer
Washing Buffer (before use, add 96-99% Ethanol as indicated on the bottle)
Elution Buffer
Binding Columns
2 ml Collection Tubes
Before start
The Lysis Buffer contains an integrated pH indicator to easily
control the optimal pH value for DNA binding. Efficient DNA
binding (for Column loading) requires a pH lower than 7.5 that is
indicated by a color change of the indicator to bright yellow.
The kit can either be used in micro-centrifuges or on vacuum
manifolds. It enables the extraction of plasmid DNA up to 10 kb
length and yields up to 20 μg DNA per preparation.
The eluted high-quality plasmid DNA is ready to use for a variety
of down-stream application. For subsequent in vitro translation
we recommend to add RNase Inhibitor or the application of an
additional spin-column or phenol-chloroform based purification
step. This avoids any risk of carry-over contamination with RNase
due to the previous neutralization step.
Preparation Procedure:
The DNA purification follows a simple binding, washing, and
eluting procedure. The optional secondary washing step
minimizes the salt content of the purification product. Before
starting, add the following components to the respective bottles:
ŸAdd the RNase A to the Neutralization Buffer and mix well.
Neutralization Buffer containing RNase A should be stored
at 4 °C.
ŸThe activity of dissolved RNase A in Neutralization Buffer may
decrease after several months and small amounts of RNA
may be co-purified. In case RNA is detected after plasmid
purification, add additional RNase A to the Neutralization
Buffer in order to enhance enzyme activities.
ŸAdd 96-99 % Ethanol (not included in the kit) to the Washing
Buffer as indicated on the bottle. Please note that the Ethanol
concentration of Washing Buffer may decrease during long
term storage resulting in a drop-down of the final DNA yield.
Mini-prep
Mini-prep
16m
16m
- Harvest the 1 ~ 3 mL of bacterial cell culture by 10000 rpm, 00:05:00 .
- Discard the supernatant media.
- Resuspend of cell pellet with remained media by vigorously vortexing or pipetting.
- Add 300 µL of Buffer S1. (Buffer S1 is Cell lysis buffer).
- Inverting the tube immediately 2 ~ 3 times.
- Do Not Vortexing and Pipetting.
Note
At this time, cell ressuspension with Buffer S1(Lysis Buffer) by vorteing or pipetting can clivage gDNA. The cleaved gDNA affects the plasmid DNA preparation step and can be isolated together with the plasmid DNA. This reduces the efficiency of plasmid DNA prep. For this reason, mix only by the inverting method after adding Buffer S1.
5m
Neutralization:
- Add 300 µL of Neutralization Buffer (containing RNase A) to sample and mix gently by inverting the tube 4-6 times (do not vortex!).
- Centrifuge at 10000 x g, Room temperature, 00:05:00 in a microcentrifuge.
Note
NOTE: The color of the binding mixture should change to bright yellow indicating a pH lower than 7.5 required for optimal DNA binding. An orange or violet color shows a pH >7.5 and indicates an inefficient DNA adsorption. In this case, it is recommended to adjust the pH of the mixture by addition of a small volume of 3 M sodium acetate, pH 5.0 before proceeding.
5m
Column Activation:
- Place a Binding Column into a 2 ml collection tube.
- Add 100 µL of Activation Buffer into the Binding Column.
- Centrifuge at 10000 x g, 00:00:30 in a micro-centrifuge.
30s
Column Loading:
- Apply the supernatant from step 2 into the activated Binding Column by decanting or pipetting.
- Centrifuge at 10000 x g, 00:00:30 .
- Discard the flow-through from the collection tube.
30s
Column Washing:
- Apply500 µL of Washing Buffer (containing Ethanol) to the Binding Column.
- Centrifuge at 10000 x g, 00:00:30 and discard the flow-through.
30s
Optional Secondary Washing:Recommended only for DNA >200 bp, if highly purified DNA (for DNA sequencing, transfectionetc.) is required.
- Add 700 µL of Washing Buffer to the Binding Column.
- Centrifuge at10000 x g, 00:00:30 and discard the flow-through.
- Centrifuge again for 10000 x g, 00:02:00 to remove residual Washing Buffer.
2m 30s
Elution
- Place the Binding Column into a clean 1.5 ml microtube (not provided in the kit).
- Add 30-50 µL Elution Buffer or dd-water to the center of the column membrane.
- Incubate for 00:01:00 at Room temperature .
- Centrifuge at 10000 x g, 00:01:00 to elute DNA.
2m