Plasmid Maxi Preparation Kit

Name: Plasmid Maxi Preparation Kit
SKU: D0026
Price: 133.30 EUR
Availability: InStock

€ 133,30

Plasmid Maxi Preparation Kit provides a rapid method for maxi preparation of high-quality plasmid DNA from E. coli cells with a centrifuge. The extracted plasmid DNA can be used directly for cell transfection, DNA sequencing, PCR, PCR-based mutagenesis, in vitro transcription, bacterial transformation, and restriction enzyme digestion, etc. Package: 20T

SKU: D0026

Category: DNA Extraction & Purification, Molecular Biology, Plasmid Extraction, Reagents

Description

Plasmid Maxi Preparation Kit provides a rapid method for maxi preparation of high-quality plasmid DNA from E. coli cells with a centrifuge. The extracted plasmid DNA can be used directly for cell transfection, DNA sequencing, PCR, PCR-based mutagenesis, in vitro transcription, bacterial transformation, and restriction enzyme digestion, etc.

Endonuclease I encoded by the endA1 gene cuts and degrades dsDNA. Wild type E. coli containing the endA1 gene is named as EndA⁺ strain while the endA1 mutant strain is named as EndA^– strain. Please refer to Table 1 for the common EndA^– and EndA⁺ strains. This kit is suitable for isolating plasmid from the commonly used EndA^– strains such as DH5α, JM109 and XL-1 blue. Although this kit can be used for isolating plasmid from EndA⁺ strains such as JM110, BL21(DE3), TG1 and HB101, the obtained plasmids are slightly contaminated with nuclease and will degrade completely when incubated in endonuclease buffer at 37℃ for 1 hour.

This kit employs a new type of ion exchange column which binds plasmid DNA at the moment when plasmids pass through the column under appropriate conditions. After the removal of RNA, proteins or other impurities by medium-salt wash, plasmid is eluted thoroughly under certain conditions, thus achieving a rapid purification of plasmid DNA. No phenol-chloroform extraction or alcohol precipitation is required. Plasmid purification from 12 samples can be completed in less than 60 minutes.

One column of this product can extract up to 500µg plasmid from 100ml of overnight E. coli culture. The isolated plasmid DNA generally has an OD260/OD280 ratio of 1.80. The plasmid amount and purity vary depending on the copy number of plasmid and the type of bacteria strains.

Packing List：

Item	Component	Quantity
D0026-1	Solution I	105ml
D0026-2	Solution II	105ml
D0026-3	Solution III	150ml
D0026-4	Solution IV	100ml (Add 150ml of absolute ethanol before the first use)
D0026-5	Solution V	60ml
D0026-6	RNase A (100mg/ml)	105µl
D0026-7	Plasmid Purification Columns and Collection Tube	20 Sets
Manual	–	1 copy

Storage Conditions：

Store at room temperature for up to 1 year.

Precautions：

Before the first use, centrifuge the RNase A provided in this kit briefly, and then add all content into Solution I. Mix well, mark the bottle, and store Solution I at 4℃ after adding RNase A.

Add 150ml of absolute ethanol to Solution IV before the first use. Mix well and mark the bottle.

At low temperatures, Solution II and Solution III may precipitate. The precipitates can be dissolved in a 37℃ water bath. Mix well before use. Over-mixing of Solution II should be avoided to prevent the generation of air bubbles.

After each use of Solution II, close the bottle immediately and tightly to avoid acidification of Solution II from CO₂ in the air.

Solution II is strongly alkaline. Solution II, Solution III and Solution IV are irritating. Please take effective measures to avoid inhalation or direct contact with skin or eyes.

All procedures including centrifugation are performed at room temperature.

The collection tube should be reused several times for one plasmid isolation. Do not discard it in the middle of isolation.

This kit is for R&D only. Not for drug, household, or other uses.

For your safety and health, please wear a lab coat and disposable gloves during the operation.

Instruction

Instructions for Use：

1. Collect 100ml of the overnight LB culture and centrifuge at 5000×g for 1 min to harvest E. coli. Discard the supernatant.
The overnight LB E. coli culture usually has an absorbance of 2-4 at 600nm. We recommend centrifuging at 5000×g for 1 minute. Prolong the centrifugation time if bacterial cells are not precipitated sufficiently. Too long or too fast centrifugation will make the cell pellet hard to be resuspended in Solution I. For the isolation of high copy plasmid, do not use more than 150ml of LB culture, while up to 200ml LB culture can be used to isolate low copy plasmid. Excess bacteria will result in insufficient lysis.
2. Resuspend the bacterial pellet in 5ml of Solution I. The bacteria should be resuspended completely by vortex vigorously or pipetting up and down until no cell clumps remain.
Note: Ensure that RNase A has been added to Solution I.
3. Add 5ml of solution II and mix thoroughly by inverting the centrifuge tube 4-6 times. Incubate at room temperature for no more than 5 minutes.
DO NOT VORTEX! Vortex will result in shearing of genomic DNA. After mixing, the solution should become transparent and viscous. If bacterial cells are not resuspended completely in step 2, clumps or flocs may still be present after 4-6 inversions. In this case, the number of inversions can be increased by 3-5 times, and then incubate at room temperature for 2-3 minutes. Do not allow the lysis reaction to proceed for more than 5 minutes. After use, close the bottle containing Solution II immediately and tightly to avoid acidification of Solution II from CO2 in the air.
4. Add 7ml of Solution III, mix immediately and thoroughly by vigorously inverting the centrifuge tube 4-6 times, during which a fluffy white material forms and the lysate becomes less viscous.
Do not vortex and do not invert the tube too many times, otherwise the quality of the obtained plasmid will be lower.
5. Centrifuge at 12,000-14,000 rpm for 10 minutes at room temperature.
While centrifuging, prepare a plasmid purification column with a collection tube, and label the column.
6. Load the supernatant from step 5 onto the plasmid purification column. Centrifuge at 12,000-14,000 rpm for 2 minutes. Discard the flowthrough and place the column back into the collection tube.
7. Add 12ml of solution IV with ethanol to the column. Centrifuge at 12,000-14,000 rpm for 2 minutes. Discard the flowthrough and place the column back into the collection tube.
8. Centrifuge at 12,000-14,000 rpm for 2 minutes to remove any residual liquid and allow ethanol to evaporate completely.
9. Place the column in a clean 50ml centrifuge tube, add 2ml of solution V to the center of the column and leave it at room temperature for 2 minutes.
Solution V can be replaced by ddH₂O or Milli-Q grade water. A longer time of incubation at room temperature can improve the elution efficiency slightly. To obtain a higher concentration of plasmid, use 1ml of Solution V to elute DNA, but the yield may decrease by 5-10%.
10. Centrifuge at 12,000-14,000 rpm for 2 minutes to elute the purified plasmid DNA. Store plasmid DNA at 4℃ for short-term storage or at -20℃ for long-term storage.
Usually, the concentration of the obtained plasmid DNA is about 0.1-0.3mg/ml. If higher concentration of plasmid is desired, the plasmid can be concentrated using the following isopropanol precipitation method or the conventional ethanol precipitation method.
a. Add 0.7 times the volume of room temperature isopropanol (e.g., 0.7ml of isopropanol to 1ml of plasmid), mix well and centrifuge at 1,2000-14,000 rpm at 4℃ for 10 minutes. Aspirate the supernatant carefully.
b. Wash the pellet with 1ml of room temperature 70% ethanol. Centrifuge at 12,000-14,000 rpm at 4℃ for 5-10 minutes, and carefully aspirate the supernatant.
c. Centrifuge at 5,000-10,000 rpm at 4℃ for 5-10 seconds and carefully aspirate all the residual liquid with a 20µl or 200µl pipette.
d. Air dry the pellet for up to 1 minute and dissolve the plasmid in an appropriate amount of solution (e.g., Solution V, 10 mM Tris-Cl pH8.5 or Milli-Q grade water).
Do not overdry, or the plasmid will be difficult to dissolve. We recommend dissolving the plasmid in weak alkaline solutions.

Table 1. EndA^– and EndA⁺ strains of E. coli.

EndA^–	EndA⁺
BJ5183	BL21 (DE3)
DH1	CJ236
DH20	HB101
DH21	JM83
DH5α	JM101
JM103	JM110
JM105	LE392
JM106	MC1061
JM107	NM522 (all NM series are EndA⁺)
JM108	NM554
JM109	P2392
MM294	PR700 (all PR series are EndA⁺)
SK1590	Q358
SK1592	RR1
SK2267	TB1
SRB	TG1
TOP10	Y1088 (all Y10 series are EndA⁺)
XL1-Blue	BMH 71-18
XLO	ES1301