Navigation Links
Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications,,,and Agarose Gels

High yields of extremely pure DNA with StrataPrep nucleic acid purification kits


Jeff Braman Scott Basehore
Stratagene Cloning Systems, Inc.


Stratagene announces a new line of DNA purification products, which provide reliable isolation of high-purity miniprep plasmid, PCR product and gel-fractionated DNAs. Purified miniprep DNA prepared with the StrataPrep plasmid miniprep kit is easily digested with restriction enzymes, is transformed with high efficiency and is accurately sequenced. With the StrataPrep PCR purification kit, PCR products are purified devoid of primers and small, nonspecific amplification products. These purified PCR products are cloned with high efficiency. Gel-isolated DNA that is purified using the StrataPrep DNA gel extraction kit is recovered with excellent yield and is efficiently cloned.

Purification of nucleic acids is a time-consuming part of many research projects. To eliminate this bottleneck, Stratagene has developed the StrataPrep nucleic acid purification kits. These products enable researchers to purify DNA from minipreps, PCR amplifications and agarose gels rapidly and with high yield and purity.

Nucleic acid purification using the StrataPrep products is based upon the principles discussed by Vogelstein and Gillespie.1 The kits utilize a special DNA-binding solid support that is manufactured in a microspin format. This format allows easy removal of contaminants by centrifugal washing, and the desired product is eluted in a small volume of buffer or water. The purified DNA is ready for a variety of applications.

Figure 1
Purification Protocols

StrataPrep Plasmid Miniprep Kit

  • Spin 1.5-ml overnight culture
  • Lyse cells
  • Spin
  • Transfer lysate to microspin cup, spin, wash and spin
  • Add elution buffer and spin to collect plasmid DNA

StrataPrep PCR Purification Kit

  • Add DNA binding reagent and transfer to microspin cup
  • Spin
  • Wash and spin
  • Add elution and spin to collect PCR Product

StrataPrep PCR Purification Kit

  • Add gel dissolution buffer to gel slice
  • Transfer to microspin cup
  • Spin
  • Wash and spin
  • Add elution buffer and spin to collect DNA

Figure 1 outlines the simple and rapid protocols used for purifying miniprep plasmid DNA, PCR products and agarose gel-fractionated DNA. Each microspin cup in the StrataPrep plasmid miniprep kit yields up to 20 g of purified plasmid DNA from overnight bacterial cultures of 1.5 to 3 ml. Each microspin cup in the StrataPrep PCR purification kit and DNA gel extraction kit yields as much as 10 g of purified DNA.

Plasmid Purification

figure 2

Plasmid DNA isolated with the StrataPrep plasmid miniprep kit exhibits OD260 -to-OD280 ratios between 1.8 and 2.0, indicating a high degree of nucleic acid purity.2 Starting with 30 individual 1.5- to 3-ml liquid cultures, purified plasmid DNA can be obtained in an average of 2.5 minutes per miniprep. Plasmid and cosmid DNAs isolated with the StrataPrep plasmid miniprep kit are easily digested with restriction enzymes (figure 2). Plasmid DNA purified with the StrataPrep kit transforms chemically competent E. coli with efficiencies equivalent to those obtained for DNA that has been purified using cesium chloride density gradients. For example, 100 pg of a 2.9-kb plasmid purified using either the StrataPrep kit or the cesium chloride density gradient method transformed Epicurian Coli XL2-Blue ultracompetent cells with an efficiency of 2 x 109 colony forming units (cfu)/g of plasmid DNA (data not shown).

figure 3

Sequence analysis of purified nucleic acids is a rigorous functional test of DNA purity. DNA purified with the StrataPrep kit was sequenced using fluorescent dye terminator and dye primer chemistries with an ABI PRISM 377 sequencer (figure 3). In this data set, only 3 sequence ambiguities were identified out of approximately 600 bases. In a separate sequencing experiment, plasmid DNA was sequenced using a LI-COR 4000L DNA sequencer and infrared-labeled sequencing primers. Sequence comparisons were made of plasmid DNA purified using either the StrataPrep kit or a silica-gel membrane method. Both sequences were aligned with a published data set. For the silica-gel membrane method, 12 ambiguous base calls were identified in 801 bases of sequence. Only 8 ambiguities were seen in 795 bases of sequence from plasmid that was purified using the StrataPrep plasmid miniprep kit (data available upon request).

Purification of PCR Products

Figure 4

The StrataPrep PCR purification kit is effective at removing primers and nonspecific amplification products smaller than 100 bp. Figure 4 shows that ethidium bromide-stained material smaller than 100 bp is eliminated from 0.6- and 1.5-kb PCR products using the StrataPrep kit (lanes 3 and 7, respectively), whereas the silica-gel membrane retained contaminants migrating between 72 and 118 bp (lanes 4 and 8, respectively).

figure 5

Cloning efficiency of the 1.5-kb PCR product purified using the StrataPrep kit was compared to PCR product purified using the silica-gel membrane method (figure 5). A 1.5-kb PCR product was generated using amplification primers, each containing a different restriction site sequence. The PCR product was digested with both restriction enzymes, purified using each method, ligated to digested pBluescript SK(+) vector and transformed into XL2-Blue ultracompetent cells. In five separate experiments, the 1.5-kb PCR product purified using the StrataPrep PCR purification kit and cloned showed an average transformation efficiency of 9.7 x 106 cfu/g of vector DNA. The percent of clones that contained inserts averaged 90%. For the 1.5-kb PCR product purified and cloned using the silica-gel membrane method, the transformation efficiency was 5.5 x 106 cfu/g of vector DNA, and the percent of clones that contained inserts averaged 82%. Thus, purification using the StrataPrep kit resulted in nearly a 2-fold increase in cloning efficiency and a higher percent of clones with inserts.

Gel Isolation

Figure 6

Figure 6 shows the yield of DNA fragments that were recovered from a conventional 1% agarose gel using either the StrataPrep DNA gel extraction kit or the silica-gel-membrane method. The two methods appear to be equivalent for fragments ranging in size from 0.25 to 9 kb. A higher yield of the 23-kb fragment is obtained with the StrataPrep DNA gel extraction kit. Transformation efficiencies for ligated 1.5- and 0.6-kb PCR products that were isolated with the StrataPrep DNA gel extraction kit were 1.7 and 9.8 x 106 cfu/g, respectively. These values were equivalent to 1.5- and 0.6-kb PCR products purified using the silica-gel membrane method (data not shown). For PCR products that were purified using the StrataPrep DNA gel extraction kit, the percentage of clones with inserts (based on blue-white color selection) was 97% for the 1.5-kb fragment and 95% for the 0.6-kb fragment. The corresponding percent of clones with inserts for PCR products that were purified using the silica-gel membrane was 83% for the 1.5-kb PCR product and 92% for the 0.6-kb PCR product.

Conclusions

The StrataPrep plasmid miniprep, PCR purification and DNA gel extraction kits are reliable, simple and convenient to use. Excellent yield of high-purity DNA with these kits facilitates digestion, sequencing, cloning and other manipulations of DNA.

REFERENCES

  1. Vogelstein, B., and Gillespie, D. (1979) Proc. Nat. Acad. Sci. USA 76: 615-619.

  2. Sambrook, J., Fritsch, E.F., and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.


'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
2. A Simple and Rapid Technique to Purify High-Quality Midiprep DNA
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. High-Level Protein Expression, One-Column Purification, and FLAG Epitope Tagging in E. coli
Post Your Comments:
(Date:4/24/2015)... Diego, CA (PRWEB) April 24, 2015 ... Diego and other San Diego area universities, including UCSD ... 28 to judges from prominent local, national and nonprofit ... funding in the 5th Annual Social Innovation Challenge ... have proposed unique social venture ideas addressing a diverse ...
(Date:4/24/2015)... /PRNewswire/ - BioAmber Inc. (NYSE: BIOA ) has ... of bio-succinic acid used to produce a new ... that Bayer MaterialScience has begun commercializing.    ... eco, a series of bio-based waterborne polyurethane dispersions ... 65%.  IMPRANIL ® eco has been developed ...
(Date:4/24/2015)... 2015 PAREXEL International Corporation (NASDAQ: ... announced that Partnerships in Clinical Trials named PAREXEL,s ... Clinical Innovator of the Year.  The honor recognizes Mr. ... Company,s Perceptive MyTrials ® Data-Driven Monitoring solution. ... the 2015 Partnership Awards in Boston ...
(Date:4/23/2015)... 2015 The Thomas Brain Health ... a groundbreaking pilot study to see if early ... as little as three to six months. , ... nutritional medicine—will aggressively target and simultaneously treat the ... aging brain. These include oxidative stress, neuroinflammation, mitochondrial ...
Breaking Biology Technology:5th Annual Social Innovation Challenge at University of San Diego - Students Pitch to Live Judging Panel to Take Home $75K 25th Annual Social Innovation Challenge at University of San Diego - Students Pitch to Live Judging Panel to Take Home $75K 3Bayer MaterialScience Names BioAmber As Its Supplier For New Product Line - Bio-succinic acid forms backbone of innovative new bio-based polyurethanes for textiles 2Bayer MaterialScience Names BioAmber As Its Supplier For New Product Line - Bio-succinic acid forms backbone of innovative new bio-based polyurethanes for textiles 3Bayer MaterialScience Names BioAmber As Its Supplier For New Product Line - Bio-succinic acid forms backbone of innovative new bio-based polyurethanes for textiles 4PAREXEL Honored With 2015 Clinical Innovator Of The Year Award From Partnerships In Clinical Trials 2PAREXEL Honored With 2015 Clinical Innovator Of The Year Award From Partnerships In Clinical Trials 3PAREXEL Honored With 2015 Clinical Innovator Of The Year Award From Partnerships In Clinical Trials 4PAREXEL Honored With 2015 Clinical Innovator Of The Year Award From Partnerships In Clinical Trials 5New Study Looks to Reverse Alzheimer’s Disease 2New Study Looks to Reverse Alzheimer’s Disease 3
... developed a new test to detect the levels of vitamin ... simpler diagnosis that could help to avoid the potentially fatal ... today, 23 June 2011, in IOP Publishing,s Journal of ... breath test to more accurately measure vitamin B12. ...
... 2011 Clinical Research Management, (ClinicalRM) a full-service ... applied research, clinical trials, and regulatory support, announced ... $97MM research support contract titled Support for Military ... performed at the Walter Reed Army Institute of ...
... 22, 2011 CMC Biologics today announced that it ... steel biopharmaceutical production facility at its Bothell campus and ... new logo and a compelling tagline. These recent changes ... (Logo:   http://photos.prnewswire.com/prnh/20110502/SF93356LOGO ) The ...
Cached Biology Technology:A breath of fresh air for detecting vitamin B12 deficiency 2ClinicalRM (formerly CRM) Awarded $97MM U.S. Army Contract to Support Military Clinical Research for the Warfighter 2CMC Biologics Expands Its Production Facility and Rebrands Its Corporate Identity 2
(Date:4/2/2015)... 2015 At its 2015 ACMG Annual Clinical ... , the American College of Medical Genetics and Genomics ... its Board.  Members of the ACMG Board of Directors ... and advancing its policies and programs. ACMG is the ... "It,s an eventful time in medical genetics and genomics.  ...
(Date:3/31/2015)... , March 31, 2015  Elephant Talk Communications ... a global provider of Software Defined Network Architecture (ET ... announced total revenue of approximately $20.4 million for the ... restated $19.5 million for the year ended December 31, ... 2014, the Company had completed its multi-year transition away ...
(Date:3/24/2015)... , Mar. 24, 2015 Research and Markets ( ... Voice Biometrics - Global Strategic Business Report" report to their ... and Voice Biometrics in US$ Thousands by the following Segments: Face ... the US, Canada , Japan ... Middle East & Africa ...
Breaking Biology News(10 mins):American College of Medical Genetics and Genomics Announces New Board Members: Dr. Louanne Hudgins is ACMG President-Elect 2American College of Medical Genetics and Genomics Announces New Board Members: Dr. Louanne Hudgins is ACMG President-Elect 3American College of Medical Genetics and Genomics Announces New Board Members: Dr. Louanne Hudgins is ACMG President-Elect 4American College of Medical Genetics and Genomics Announces New Board Members: Dr. Louanne Hudgins is ACMG President-Elect 5American College of Medical Genetics and Genomics Announces New Board Members: Dr. Louanne Hudgins is ACMG President-Elect 6American College of Medical Genetics and Genomics Announces New Board Members: Dr. Louanne Hudgins is ACMG President-Elect 7American College of Medical Genetics and Genomics Announces New Board Members: Dr. Louanne Hudgins is ACMG President-Elect 8Elephant Talk Reports 2014 Fourth Quarter and Year End Financial Results 2Elephant Talk Reports 2014 Fourth Quarter and Year End Financial Results 3Elephant Talk Reports 2014 Fourth Quarter and Year End Financial Results 4Elephant Talk Reports 2014 Fourth Quarter and Year End Financial Results 5Elephant Talk Reports 2014 Fourth Quarter and Year End Financial Results 6Face and Voice Biometrics - Global Strategic Business Report 2015: Face Recognition Gains Prominence in Immigration Control 2Face and Voice Biometrics - Global Strategic Business Report 2015: Face Recognition Gains Prominence in Immigration Control 3Face and Voice Biometrics - Global Strategic Business Report 2015: Face Recognition Gains Prominence in Immigration Control 4Face and Voice Biometrics - Global Strategic Business Report 2015: Face Recognition Gains Prominence in Immigration Control 5Face and Voice Biometrics - Global Strategic Business Report 2015: Face Recognition Gains Prominence in Immigration Control 6
... 2007 Men who have lower-pitched voices have more ... found. And their study suggests that for reproductive-minded ... The study, published in Biology Letters, offers insight into ... how we choose our mates. In previous studies, ...
... that could improve a class of drugs based on antibodies, ... structural changes in an antibody that will improve its effectiveness. ... create a new version of cetuximab, a drug commonly used ... 10 times greater affinity than the original molecule. The ...
... Legacy of Regulatory Submissions Leadership Strengthened with Clinical ... Trials Services and Standards-Based Data Management, ... Solutions,Inc. (ISI) announced today it agreed to terms ... Sciences industry moving to,standards-based regulatory submissions, the acquisition ...
Cached Biology News:MIT model could improve some drugs' effectiveness 2MIT model could improve some drugs' effectiveness 3Image Solutions, Inc. Acquires Zurich Biostatistics, Inc. 2Image Solutions, Inc. Acquires Zurich Biostatistics, Inc. 3
... polyclonal to FbxL7 The F ... which it was originally observed, is ... binds SKP1. F-box proteins are components ... called SCFs (SKP1, cullin, F-box proteins), ...
Rabbit polyclonal to SERHL ( Abpromise for all tested applications). entrezGeneID: 94009 SwissProtID: Q9H4I8...
Sheep polyclonal to Staphylococcus DNase ( Abpromise for all tested applications). Antigen: Full length native protein (purified)....
Allophycocyanin (APC) anti-human CD193 (CCR3, CKR3) 25 tests...
Biology Products: