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EasyPrep HY-Midi Plasmid
Extraction Kit
For purification of high pure plasmid DNA with high yields
EasyPrep HY-Midi Plasmid
Extraction Kit
Cat No. DPT-BA16
Kit Contents
Contents
DPT-BA16
10 preps
RNase A (100 mg/ml)
500 µl
Buffer P1
100 ml
Buffer P2
100 ml
Buffer P4
100 ml
Buffer TB
30 ml
EasyRed
500 μl
Filtration Columns CS1
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Storage
EasyPrep HY-Midi Plasmid Extraction Kit can be stored dry at room temperature (15-25°C) for up to
12 months without showing any reduction in performance and quality. For longer storage, it can be
stored at 2-8°C. If any precipitate forms in the buffers after storage at 2-8°C, it should be dissolved by
warming the buffers to 37°C before use. RNaseA (100mg/ml) can be stored for one year at room
temperature (15-25°C). After adding RNaseA, Buffer P1 is stable for 6 months at 2-8°C.
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Introduction
EasyPrep HY-Midi Plasmid Extraction Kit adopts specially designed buffer system that is used in
conjunction with isopropanol precipitation to achieve fast and efficient purification of high pure
plasmid DNA. Plasmid DNA prepared by this Kit is suitable for a variety of routine applications
including restriction enzyme digestion, sequencing, library screening, ligation and transformation, in
vitro translation, and transfection of cell lines. Recommended bacterial culture volume: 500-1500 ug
plasmid with 100 ml bacterial culture for high-copy vectors; and 200-400 ug plasmid with 200 ml
bacterial culture for low- copy vectors.
Using EasyRed reagent
EasyRed reagent is a color indicator which provides visual identification of optimum buffer mixing.
This prevents common handling errors that lead to inefficient cell lysis and incomplete precipitation of
SDS, genomic DNA, and cell debris. Researchers can determine whether to use EasyRed according to
their experience and experiment purpose. EasyRed has no influence on following reactions, such as
PCR, enzyme digestion and sequencing. If the purified plasmid is to be used in transfection experiment,
however, usage of EasyRed is not recommended. EasyRed should be added to the Buffer P1 before use
at a ratio of 1:200. Mix thoroughly by inverting, and the solution will present transparent red.
The plasmid preparation procedure is performed as usual. After add Buffer P1 to harvested bacterial
cells, the suspension will become cloudy. After addition of Buffer P2 to Buffer P1 the color of the
suspension changes to purple. Mixing should result in a homogeneously transparent purple suspension,
showing the lysis is complete. Upon addition of Buffer P4, EasyRed turns transparent yellow. The
presence of a homogeneous solution with no traces of purple indicates that SDS from the lysis buffer
(Buffer P2) has been effectively precipitated.
Important Notes
1. Prevent to contact Buffer P2 and P4 directly. Close the bottle containing Buffer P2 and P4
immediately after use to avoid acidification of Buffer P2 and P4 from CO2 in the air.
2. Draw out the plunger from the filter slowly to avoid membrane loose.
3. The amount of extracted plasmid is related to cells concentration and plasmid copy. If working with
low copy vectors or large plasmid (>10 kb), it may be beneficial to increase culture volume and to
increase Buffer P1, P2, and P4 in proportion. Warm the Buffer TB to 50-60°C before use. Prolong
adsorption and elution time properly to increase extraction efficiency.
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Things to do before starting
1. Add the provided RNase A solution (500 μl) to Buffer P1, mix, and store at 2-8°C.
2. Check Buffer P2 and P4 for SDS precipitation due to low storage tempertures. Redissolve the
precipitate by warming to 37°C. Do not shake Buffer P2 vigorously.
3. Prepare 60 ml 5 M NaCl solution.
4. Optional: Add the provided EasyRed reagent to Buffer P1 before use at a ratio of 1:200. If the
purified plasmid is to be used in transfection experiment, however, usage of EasyRed is not
recommended.
Protocol
1. Harvest 100 ml bacterial culture cells by centrifuging at 10,000 rpm (~11,500 × g) for 3 min at room
temperature (15-25°C), and then remove all traces of supernatant by inverting the open centrifuge tube
until all medium has been drained.
2. Resuspend pelleted bacterial cells in 10 ml Buffer P1.
Note: Ensure that RNase A has been added to Buffer P1. The bacteria should be resuspended
completely by vortexing or pipetting up and down until no cell clumps remain. If EasyRed
reagent has been added to Buffer P1, mix thoroughly before use. The suspension become cloudy
red due to the presence of bacteria cells.
3. Add 10 ml Buffer P2 and mix thoroughly by inverting the tube 6-8 times, and incubating at room
temperature for 5 min.
Note: Mix by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA.
If necessary, continue inverting the tube until the solution becomes viscous and slightly clear.
Do not allow the lysis reaction to proceed for more than 5 min.
If EasyRed has been added to Buffer P1 the cell suspension will turn purple after addition of
Buffer P2. Mixing should result in a homogeneously colored suspension. If the suspension
contains localized red regions or if brownish cell clumps are still visible, continue mixing the
solution until a homogeneously colored suspension is achieved.
4. Add 10 ml Buffer P4, and mix immediately and thoroughly by vigorously inverting 6-8 times, until
the whole solution become cloudy. Incubate at room temperature for 10 min. A white fluffy material
forms.
Note: To avoid localized precipitation, mix the lysate thoroughly and immediately after addition
of Buffer P4. Mix by inverting the tube. Do not vortex.
If EasyRed has been used, the suspension should be mixed until all trace of purple has gone and
the suspension become transparent yellow. A homogeneous yellow suspension indicates that the
SDS has been effectively precipitated.
5. Centrifuge for 10 min at 10,000 rpm (~11,500 × g). The white material should be precipitated to the
bottom of the centrifuge tube and form a white pellet.
Note: The centrifugation time can be prolonged appropriately to precipitate the white material.
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If use more than 100 ml bacterial culture, prolong centrifugal time to 20-30 min.
6. Transfer the supernatant into a Filtration Column CS1. Gently insert the plunger into the Filtration
Column CS1 and filter the cell lysate into a new 50 ml tube (not supplied in the kit).
Note: If the white material fails to be precipitated to the bottom in step 5, it means the lysate had
not been mixed thoroughly in step 5. In this scenario, avoid transferring large clump into the
Filtration Column CS1, which will clog the filtration membrane. Small fragments have on
influence on Filtration Column CS1.
7. Add 0.35 volume isopropanol to the cleared lysate, and then add 5M NaCl of half amount of the
isopropanol. Sealed the received tube, and mix thoroughly by inverting the tube.
Note: it is a normal phenomenon if no visible precipitate appears in this step.
8. Centrifuge at 10,000 rpm (~11,500 × g) for 30 min at 4°C. Decant all the supernatant.
9. Wash the pellet with 6 ml 70% ethanol. Centrifuge at 10,000 rpm (~11,500 × g) for 10 min at 4°C.
Decant all the supernatant without disturbing the pellet.
10.
Repeat the step 9.
11. Air dry the plasmid DNA pellet for 10-20 minutes. And redissolve the DNA in 1-1.5 ml Buffer TB.
Note: Over-drying the pellet will make the DNA difficult to redissolve. DNA dissolves best
under slightly alkaline conditions. Buffer TB or distilled water (pH 7.0-8.5) is recommended to be
used to redissolve the plasmid DNA. For long-term storage of DNA, dissolving in Buffer TB and
storing at –20°C can avoid the possible acid hydrolysis of DNA in water.
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