Navigation Links
A Simple and Rapid Technique to Purify High-Quality Midiprep DNA


High-quality plasmid DNA for all demanding molecular biology applications

Scott Basehore Jeff Braman
Stratagene

A new technique is now available to quickly, easily, and reliably isolate high-purity plasmid DNA. The convenient spin-format eliminates the need for resin suspensions, gravity flow columns, and alcohol precipitations, while easily accommodating simultaneous multiple purifications. The purified DNA is ready for all applications, including sensitive automated sequencing and mammalian cell transfection.

StrataPrep midiprep kit purification of plasmid DNA1 is based on alkaline lysis of bacteria and binding of the liberated plasmid DNA to a glass fiber matrix contained in a midispin cup. Washing the spin cup removes contaminants, and the DNA is eluted in a small volume of buffer. The new midispin cup design and specially formulated alkaline lysis reagents allow up to 350 g of plasmid DNA to be recovered from a single device. The resulting DNA can be used for applications such as restriction digestion, cloning, PCR, site-directed mutagenesis, manual and automated fluorescent sequencing, and mammalian transfection.

Endotoxin, prevalent in plasmid preparations, is difficult to separate from plasmid DNA and results in toxicity to cultured mammalian cells.2 The StrataPrep EF midiprep protocol provides an optional extraction of endotoxins, which are reduced to 0.1 to 0.2 EU/g DNA (endotoxin units per microgram of plasmid DNA); hence, high-efficiency transfection results.

Fast and Easy Method

Fig.1

The StrataPrep midiprep procedure includes a unique color-indicating method that facilitates successful DNA purification (Figure 1). A modified alkaline lysis and centrifugation produces a cleared bacterial lysate. A chaotropic salt is added to the lysate, and the mixture is transferred to a midispin cup. Upon centrifugation, plasmid DNA binds to the glass fiber matrix in the midispin cup. Contaminants are removed by washing the midispin cup, and the plasmid DNA is eluted in a small volume of buffer. No alcohol precipitation is necessary to concentrate the DNA. Endotoxin is efficiently removed by optionally extracting the cleared bacterial lysate prior to adding the chaotropic salt. In one hour, the standard midiprep plasmid purification is complete (only 1.5 hours with endotoxin removal); the standard anion exchange method takes a minimum of 3 hours to complete.

High Yields and Purity

Fig.2

The maximum binding capacity of a StrataPrep midispin cup is 350 g of plasmid DNA (Figure 2). The binding capacity of a single StrataPrep midispin cup was determined by loading various known quantities of purified pBluescript plasmid into midispin cups, then washing and eluting the DNA. The quantity of eluted DNA was measured by UV spectrophotometry. Nearly 350 g of high-copy number pBluescript II SK(+) plasmid DNA was also recovered from a 100-ml overnight bacterial culture using a single midispin cup, while the low-copy number pCMV gal plasmid yield from a 200-ml overnight culture was 250 g (Figure 3). Yields and spectrophotometric values (A260/A280 nm) between 1.8 and 2.0 were identical, using both the standard and endotoxin-free protocol (data not shown).

Figure 3

Quality Automated Sequencing

The quality of StrataPrep midiprep plasmid DNA was tested in automated fluorescent sequencing. Plasmid DNA purified by a StrataPrep standard midiprep, a StrataPrep endotoxin-free midiprep, twice-banded CsCl density gradient centrifugation, and anion exchange methods were sequenced at Sequetech Corporation. High-signal, low-background sequence data was obtained with all four plasmid preparations. Figure 4 shows representative sequence data for StrataPrep endotoxin-free midiprep purified DNA. Sequence read length for the four templates was approximately 680 bases. There were a number of sequence ambiguities generated from each template, starting arbitrarily from nucleotide 20: one for a StrataPrep midiprep standard, one for a StrataPrep midiprep endotoxin-free, zero for CsCl density gradient centrifugation, and four for anion exchange.

Figure 4

Increased Transfection Efficiency

Endotoxins are toxic to mammalian cell cultures and can greatly affect transfection efficiencies. The StrataPrep midiprep endotoxin-free extraction protocol reduces endotoxin levels to between 0.1 to 0.2 EU/g plasmid DNA. LipoTAXI transfection reagent3,4 and calcium phosphate5 reagent methods were used to transfect StrataPrep endotoxin-free purified DNA into Cos-7, DU 145, and JAR cell lines and resulted in higher transfection efficiencies, compared to anion exchange enndotoxin-free purified plasmid DNA (Figure 5).

Figure 5

Supplemental Components

Endonuclease A removal buffer is available separately when purifying plasmid DNA from End A+ bacterial strains such as JM101. A simple wash step removes endonucleases from the preparation, eliminating non-specific degradation of the plasmid DNA due to the bacterial strain used.

Pre-qualified pyrogen-free 50-ml conical tubes are also available for the preparation of endotoxin-free DNA for sensitive experiments. New plasticware is also considered endotoxin-free.

Conclusions

Use the fast, easy StrataPrep EF plasmid midiprep kit to isolate high-purity endotoxin-free plasmid DNA without resin slurries, toxic chemicals, or specialized equipment. With its convenient format, perform multiple preparations simultaneously, and generate yields similar to maxiprep scale purification. StrataPrep midiprep purified DNA results in accurate automated fluorescent sequence data with high intensity and low background, and the endotoxin-free DNA is transfected into mammalian cells with high efficiency.

REFERENCES
  1. Braman, J. and Basehore, S. (1997) Strategies 10: 84-86.

  2. Cotton, M., et al. (1994) Gene Therapy 1: 239-246.

  3. Pingerelli, P., Petre, M., and Karmiol, S. (1997) Strategies 10: 8-9.

  4. Stratagenes LipoTAXI Transfection Reagent Manual.

  5. Spector, D.L., Goldman, R.D., and Leinwand, L.A. (1998) In Cells: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. p. 86.2.


'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
2. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
3. Simple, Sensitive Isotyping of Mouse Monoclonal Antibodies
4. Simple Isolation of RNA from Tissue and Cultured Cells
5. HSVision Molecular Beacon Detection Module Rapidly Detects Herpes Simplex Virus DNA
6. Simple and Rapid Isolation of Plant RNA Using the Eppendorf Perfect RNA, Eukaryotic, Maxi Kit
7. The Fastest, Simplest, and Most Effective Way to Remove DNA Contamination
8. Simple Approaches for Optimization of RT-PCR
9. Detection of Morphine and Morphine-Glucuronide in Saliva or Urine with a Rapid and Simple Method Without Derivatization on the Varian 500-MS
10. Gram Quantities of MAb Produced with Simple Bioreactor in Serum-Free Perfusion Culture Replacing Ascitic Fluid Production
11. CastAway Precast Gels for Rapid Automated DNA Sequence Analysis
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Simple and Rapid Technique Purify High Quality Midiprep DNA

(Date:7/11/2014)... Research and Markets  has announced the ... Global Industry Analysis, Size, Share, Growth, Trends and Forecast ... http://photos.prnewswire.com/prnh/20130307/600769 Transfection is ... or RNA) into cells. This technology helps the cells ... protein metabolism by affecting the nuclear genes. The transfection ...
(Date:7/11/2014)... July 11, 2014 Cayenne Medical, ... specializing in the soft tissue reconstruction segment, announced ... System for surgeries involving the shoulder and extremities. ... a novel, inserter-controlled deployment method. The unique delivery ... deployment, anchor pull-out, or anchor displacement (also commonly ...
(Date:7/10/2014)... Terascala, the industry leader in ... a former vice president of product management for Flexera ... of marketing. Terascala’s software when combined with leading compute ... highest performance and most reliable solutions for processing big ... channel expansion and broaden its product portfolio. His leadership ...
(Date:7/10/2014)... July 10, 2014 Senior supply chain ... professional associations, addressed the challenges of “Reducing Cost, Lead ... Industries” through strategic sourcing. Describing the partnership of the ... Supply Chain Management Institute ( SCMI) of the ... June 24 at the Kroc Institute of Peace and ...
Breaking Biology Technology:International Transfection Technology Market - Industry Analysis, Size, Share, Growth, Trends and Forecast to 2019 2Cayenne Medical Launches SureLock™ All-Suture Anchor System for Shoulder and Extremity Surgery 2Cayenne Medical Launches SureLock™ All-Suture Anchor System for Shoulder and Extremity Surgery 3Terascala Expands Executive Team 2Terascala Expands Executive Team 3Biotech & Biopharm Supply Chain Management Executives Launch BSMA in San Diego 2Biotech & Biopharm Supply Chain Management Executives Launch BSMA in San Diego 3Biotech & Biopharm Supply Chain Management Executives Launch BSMA in San Diego 4
... Fla., April 9 Cryo-Cell International, Inc. ... world,s largest and most established family cord blood banks, ... 28, 2009. Consolidated revenues for the first quarter ... approximately $4.2 million for the first quarter of fiscal ...
... the release of it,s advanced nutritional supplement, Caplex, ... a unique patent pending formulation created by ... provides cancer patients a comprehensive source of the ... on results accumulated from medical research. Impressive changes ...
... NANTONG, China, April 8 Sinobiopharma Pharmaceutical Group ... the appointment of Ms. Yan Tan as Director ... Co., Ltd., Netherlands. She served as a China-based ... sold to Schering-Plough. Prior to Organon, Ms. Tan ...
Cached Biology Technology:Cryo-Cell International, Inc. Reports First Quarter 2009 Results 2Cryo-Cell International, Inc. Reports First Quarter 2009 Results 3Cryo-Cell International, Inc. Reports First Quarter 2009 Results 4Cancer Fighting Nutrition Supplement, Caplex, Offers Cancer Patients A New All-Natural Treatment Option 2Sinobiopharma, Inc. Appoints New Director of Marketing 2
(Date:7/11/2014)... Shenzhen, July 10, 2014---A team of researchers from The ... have identified a gene of wild soybean linked to ... to grow in saline soil. This study published online ... to unveil novel genomic information for crop improvement. , ... to domestication and human selection, cultivated soybeans have less ...
(Date:7/11/2014)... Researchers have pioneered a revolutionary new way ... called Virtual Finger, allows scientists to move through ... synapses using the flat surface of their computer ... studies orders of magnitude more efficient, saving time, ... many areas of experimental biology. The software and ...
(Date:7/11/2014)... Evidence exists that cannabinoid receptor type 1 ... Ca2+ influx, and reduce neurotransmitter release. However, ... 1 can increase extracellular Ca2+ influx and ... his team, Tongji Hospital Affiliated to Tongji ... Technology, China used whole cell voltage-clamp and ...
Breaking Biology News(10 mins):BGI reports a novel gene for salt tolerance found in wild soybean 2Virtual finger enables scientists to navigate and analyze complex 3D images 2
... Of Southern California School Of Dentistry have uncovered another clue ... leads to palate formation. Cleft palate is one ... out of every 700 live births. Clefts are more common ... males are twice as likely to have a cleft lip, ...
... record biometric revenues and profitability, WALL, N.J., ... BKYI), a leader in wireless public safety and,finger-based ... and strong revenue growth for its second quarter,ended ... operations for the quarter ended June 30,2008 was ...
... As people age, their cells become less efficient at ... of toxic material that is especially pronounced in Alzheimer,s, Parkinson,s ... first time, scientists at the Albert Einstein College of Medicine ... entire organ the liver and shown that, as ...
Cached Biology News:USC researchers identify alternate pathway that leads to palate development 2USC researchers identify alternate pathway that leads to palate development 3BIO-key(R) Reports Profitability on Strong Revenue Growth for Second Quarter 2008 2BIO-key(R) Reports Profitability on Strong Revenue Growth for Second Quarter 2008 3BIO-key(R) Reports Profitability on Strong Revenue Growth for Second Quarter 2008 4BIO-key(R) Reports Profitability on Strong Revenue Growth for Second Quarter 2008 5In scientific first, Einstein researchers correct decline in organ function associated with old age 2In scientific first, Einstein researchers correct decline in organ function associated with old age 3
Request Info...
Request Info...
Epi-Grids™ Colony Grid Templates are easy-to-use griding templates that allow rapid organization of colonies from initial screening plates and for preparing precise master plates....
Competitors to lower non-specific signal...
Biology Products: