Navigation Links
Purified Single-Stranded M13 DNA for Automated Sequencing


A streamlined and reliable method to obtain high-quality single-stranded DNA

Jeff Braman Scott Basehore
Stratagene

The StrataPrep M13 DNA purification kit allows easy, quick, and reliable purification of bacteriophage single-stranded DNA. The yield of single-stranded DNA recovered from phage lysate exceeds the yield obtained with the leading commercial silica-gel-based (SGB) M13 DNA purification method. The resulting high-purity DNA is ideal for automated sequencing applications.

M13 DNA is a preferred template for many sequencing projects.1-5 For many years, the standard laboratory protocol used to purify M13 DNA relied upon polyethylene glycol (PEG)-NaCl precipitation of phage particles, phenol-chloroform extraction to remove phage coat proteins, and ethanol precipitation to concentrate the DNA.6 This method is time-consuming, employs hazardous chemicals, and is difficult to apply to more than a few samples.

However, with the StrataPrep M13 DNA purification kit, isolating high-purity M13 DNA is possible without these cumbersome steps because the procedure is streamlined: Collect M13 phage particles on the unique glass fiber matrix contained in a StrataPrep spin cup;7 add a chaotropic salt solution to the spin cup so the phage coat protein denatures and binds the liberated single-stranded DNA to the glass matrix; centrifuge-wash the phage DNA to remove contaminating protein and salt; and elute the DNA from the spin cup with a low ionic strength buffer. The concentration of the highly purified DNA that results is ideal for automated sequencing applications.

High Yield Recovery of Single-Stranded DNA

The yield of single-stranded DNA from StrataPrep M13 method of Phagescript lysate (a modified M13 bacteriophage8), was at least twice that obtained from the SGB method (Figure 1). For 3 ml of phage lysate, the StrataPrep method yielded 7.5 g of DNA at a concentration of 25 ng/l, as judged by gel electrophoresis.

Figure 1

Quality Automated Sequencing

Equal volumes of StrataPrep- and SGB-purified Phagescript DNA were subjected to fluorescent dye primer automated sequencing by an independent sequencing service (Sequetech Corp.; Figure 2). Signal intensity was more than twice as high for the StrataPrep-purified template (Figure 2, Panel A) compared to the SGB-purified template (Figure 2, Panel B). Sequence read length was nearly 700 bases for both templates. There were three and four sequence ambiguities scored for the StrataPrep- and SGB-purifed templates, respectively, within the first 20 nucleotides. Sequence ambiguities also occurred at nucleotides 553 and 638 for the StrataPrep-purified template and at nucleotide 44 for the SGB-purified template.

Figure 2

Conclusion

The StrataPrep M13 DNA purification kit contains a fast, easy, and reliable method for M13 purification. Recover single-stranded DNA from phage lysate more efficiently using the StrataPrep method than with the leading commercial silica-gel-based method, and realize high-quality automated fluorescent sequencing results as well. Additionally, the kit can be used to purify single-stranded DNA from rescued phagemid clones.

REFERENCES
  1. Richards, S., et al. (1994) In Automated DNA Sequencing and Analysis (Eds. Adams, M.D., et al.) pp. 191-198. Academic Press, San Diego.
  2. Wilson, R.K. and Mardis, E.R. (1997) In Genome Analysis: A Laboratory Manual (Eds. Birren, B., et al.) Vol. 1, p. 434. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
  3. Blattner, F.G., et al. (1997) Science 277: 14531462.
  4. Ali Ansari-Lari, M., et al. (1998) Genome Res. 8: 29-40.
  5. Anderson, B., et al. (1998) Genome Res. 8: 809-816.
  6. Maniatis, T., Fritsch, E., and Sambrook, J. (1982) In Molecular Cloning: A Laboratory Manual, Second Edition. pp. 4.29-4.30. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
  7. Braman, J. and Basehore, S. (1997) Strategies 10: 84-86.
  8. Stratagene Catalog (1999) p. 65.

'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Automated Fluorescent-Tag Cycle Sequencing
2. CastAway Precast Gels for Rapid Automated DNA Sequence Analysis
3. The DIG System Nonradioactive Automated High-Throughput In Situ Hybridization: a Powerful Tool for Functional Genomics Research
4. Agilent 2100 Bioanalyzer Automated Analysis System
5. Automated RNA Amplification for Arrays
6. Quantitation of DNA for Automated Sequencing Using the VersaFluor Fluorometer
7. Performance Comparison of the Experion Automated Electrophoresis System and a Competing Automated System for Protein Analysis, Rev A
8. Total RNA Purification from Cultured Cells Using the ABI PRISM 6700 Automated Nucleic Acid Workstation and Total RNA Lysis Reagents
9. DNA Damage and Repair - Quantification of Sub-Cellular Events Using Automated Confocal Imaging
10. Small-Molecule Quantitation Using Automated Single-Shot Averaging MALDI-TOF Mass Spectrometry
11. Automated Dissolution of Prednisone with the MultiDose G3
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/22/2017)... , Feb. 22, 2017  PrimeVax Immuno-Oncology, Inc. announced ... presenting at the Annual Biocom Global Life Science Partnering ... at 11:15 AM, at the Torrey Pines Lodge, in ... the organizers at Biocom who have chosen our company, ... of biotechnology companies, investors, and clinical researchers," said Mr. ...
(Date:2/22/2017)... ... February 22, 2017 , ... ... provider of women’s health, primary care, and specialty education, announced today it ... Education (ACCME). ACCME’s Accreditation with Commendation is a six-year accreditation and is ...
(Date:2/22/2017)... ... February 22, 2017 , ... ... and scientists from around the world, is pleased to announce the 2nd annual ... 23, 2017. This premier, online-only conference focused on the development and advancements in ...
(Date:2/22/2017)... 22, 2017 Dublin ... "Global Biological Crop Protection (Bio-Pesticide) Market-By Type, By Application, By End ... to their offering. ... Global Biological Crop Protection Market is forecasted to grow at ... growth in biopesticide or biological crop protection market is driven by ...
Breaking Biology Technology:
(Date:2/7/2017)... Ind. , Feb. 7, 2017 Zimmer ... leader in musculoskeletal healthcare, will present at the LEERINK ... New York Palace Hotel on Wednesday, February 15, 2017 ... live webcast of the presentation can be accessed at ... replay following the conference via Zimmer Biomet,s Investor Relations ...
(Date:2/6/2017)... According to Acuity Market Intelligence, ongoing ... to continue to embrace biometric and digital identification ... Border Control (ABC) eGates and 1436 Automated Passport ... 163 ports of entry across the globe. Deployments ... combined CAGR of 37%. APC Kiosks reached 75% ...
(Date:2/3/2017)... A new independent identity strategy consultancy firm ... . Designed to fill a critical niche in technical ... partners Mark Crego and Janice Kephart ... identity expertise that span federal governments, the 9/11 Commission, ... combined expertise has a common theme born from a ...
Breaking Biology News(10 mins):