Navigation Links
Easy Cosmid DNA Purification with StrataPrep Kits


Miniprep and midiprep quantities of pure high-kb DNA

Jeff Braman Scott Basehore
Stratagene

The StrataPrep plasmid miniprep and midiprep kits are designed to isolate up to 20 and 350 g, respectively, of high-purity plasmid DNA from a single spin cup.1,2 These kits can also easily purify very large plasmids, including cosmid DNA.

To demonstrate cosmid purification, a SuperCos I clone containing a 40-kb insert from human chromosome 11 was isolated from two, 100-ml overnight cultures using the StrataPrep EF plasmid midiprep kit. The cosmid DNA yield from the two cultures was 45 and 51 g, respectively. The A260/A280 nm ratios were 1.8 and 1.9, and the A260/A240 nm ratios were both 1.7, indicating an absence of protein, SDS, and EDTA contaminants.3 Contaminating chromosomal DNA was also absent. The resulting cosmid DNA was digested to completion with EcoR I (Figure 1).

Fig.1

Cosmid DNA can be also be isolated from 1.5-ml overnight cultures using the StrataPrep miniprep kit. The cosmid DNA is completely digested with restriction enzymes such as EcoR I (Figure 2).1

Fig.2

The midiprep purified cosmid DNA was subjected to automated fluorescent sequencing, a very sensitive application demanding pure template DNA. A representation of the high-quality sequence is shown in Figure 3. The read length was approximately 630 bases with only four sequence ambiguities.

Fig.3

The StrataPrep plasmid miniprep and midiprep kits both produce high yields of all sizes of DNA from 2.9-kb plasmids to 45-kb cosmids. Both have rapid protocols using a modified alkaline lysis method to yield quality sequence with automated or manual methods. The amount of purified DNA required determines which kit to use. StrataPrep purified cosmid DNA can serve as the source material for subclone library construction used in shotgun sequencing projects.4

REFERENCES
  1. Basehore, S. and Braman, J. (1998) Strategies 12: 5-7.
  2. Braman, J. and Basehore, S. (1997) Strategies 10: 84-86.
  3. Held, P. (1998) Pharmaceutical Laboratory, February issue.
  4. Wilson, R. K. and Mardis, E. (1997) In B. Birren, et al (Eds.), Genome Analysis, A Laboratory Manual. pp. 397-403, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

'"/>

Source:


Page: All 1 2

Related biology technology :

1. Isolation of Cosmids using Eppendorfs Perfectprep Plasmid Mini Kit
2. High-Level Protein Expression, One-Column Purification, and FLAG Epitope Tagging in E. coli
3. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
4. Yeast Protein Production System Features High Yields and One-Step Purification
5. Expression and Purification of Recombinant Proteins That Have Native Amino Acid Sequence
6. MinElute PCR Purification Kit
7. QIAquick PCR Purification Kit
8. Microarray Target Purification Kit and the Microarray Target Synthesis Kit (T7)
9. Double-Affinity Purification of GST-His Tagged Proteins Using Vivapure 8-to-96 Well Cobalt-Chelate Kit
10. Ultra-Fast Purification of Small Nucleic Acids
11. RNAClean - RNA & cDNA in vitro Reaction Purification
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/23/2016)... 2016 /PRNewswire/ - FACIT has announced the creation ... biotechnology company, Propellon Therapeutics Inc. ("Propellon" or "the ... a portfolio of first-in-class WDR5 inhibitors for the ... WDR5 represent an exciting class of therapies, possessing ... for cancer patients. Substantial advances have been achieved ...
(Date:6/23/2016)...  The Biodesign Challenge (BDC), a university competition that ... living systems and biotechnology, announced its winning teams at ... New York City . The teams, ... at MoMA,s Celeste Bartos Theater during the daylong summit. ... curator of architecture and design, and Suzanne Lee ...
(Date:6/23/2016)... June 23, 2016 Apellis Pharmaceuticals, Inc. ... clinical trials of its complement C3 inhibitor, APL-2. ... multiple ascending dose studies designed to assess the ... subcutaneous injection in healthy adult volunteers. ... as a single dose (ranging from 45 to ...
(Date:6/23/2016)... ... June 23, 2016 , ... ... and technical consulting, provides a free webinar on Performing Quality Investigations: ... 2016 at 12pm CT at no charge. , Incomplete investigations are still a ...
Breaking Biology Technology:
(Date:5/16/2016)... YORK , May 16, 2016   EyeLock ... solutions, today announced the opening of an IoT Center ... to strengthen and expand the development of embedded iris ... an unprecedented level of convenience and security with unmatched ... authenticate one,s identity aside from DNA. EyeLock,s platform uses ...
(Date:5/9/2016)... UAE, May 9, 2016 Elevay ... comes to expanding freedom for high net worth professionals ... in today,s globally connected world, there is still no ... could ever duplicate sealing your deal with a firm ... passports by taking advantage of citizenship via investment programs ...
(Date:4/28/2016)... and BANGALORE, India , April 28, 2016 ... a product subsidiary of Infosys (NYSE: INFY ), ... a global partnership that will provide end customers ... mobile banking and payment services.      (Logo: ... innovation area for financial services, but it also plays a ...
Breaking Biology News(10 mins):