Navigation Links
Efficiently Insert Unique Restriction Sites into Plasmid Vectors


Perform insertion mutagenesis in less than one day

Lisa Breister Jeff Braman
Stratagene

A 28-base insertion was introduced into a plasmid vector using the QuikChange site-directed mutagenesis kit.* The insertion created three unique restriction enzyme digestion sites. Plasmid DNA isolated from 9 out of 12 randomly chosen transformed clones were digested with all 3 restriction enzymes, as judged by gel electrophoresis.

With Stratagenes QuikChange kit,1 performing site-directed mutagenesis is simple, accurate, and efficient. This method permits single and multiple bases to be easily deleted and inserted into any double-stranded plasmid vector, eliminating the need for single-stranded DNA rescue and subcloning into specialized vectors. The kit relies on high-fidelity PfuTurbo DNA polymerase,** which accurately replicates plasmid DNA, thereby minimizing second-site mutations.2 These features make the QuikChange procedure the method of choice for most mutagenesis projects.

Here we describe insertion mutagenesis of a plasmid vector, which resulted in the creation of three unique restriction enzyme recognition sites.

Site-Directed Mutagenesis

To introduce restriction enzyme recognition sites into the plasmid vector, homologous oligonucleotides (49 bases long) were constructed (Figure 1). The oligonucleotides contained ten, 5 terminal bases and eleven, 3 terminal bases homologous to the 6-kb vector. The 28 interior nucleotides destined for insertion into the vector included restriction enzyme recognition sites for Bgl II, Pst I, and Xho I.

Fig.1

Modified pBK388 ve
'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Unique DNA Polymerase Formulation Excels in a Broad Range of PCR Applications
2. Unique Enhanced DNA Polymerase Delivers High Fidelity and Great PCR Performance
3. A Unique Dimension in Biological Purity
4. Unique Histochemical Substrate Permits Dual Staining In Animal Tissue
5. Unique Surface Treatment of Novel TSK-GEL ODS Columns Provide Separation of Aqueous and Fat Soluble Vitamins
6. Restriction Endonucleases Undergo Rigorous QC Testing to Ensure Optimal Performance
7. Restriction Digests of DNA Isolated with the Perfectprep Gel Cleanup Kit
8. Detection and Identification of Phosphorylation Sites in Proteins Using LC/MS/MS with Neutral Fragment Loss Mapping
9. High-Efficiency Site-Directed Mutagenesis of Large Plasmid Vectors
10. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
11. Generate High-Quality, Directional Plasmid cDNA Libraries
Post Your Comments:
(Date:7/30/2014)... 2014 A study presented at the ... efficacy of CSL Behring,s C1 Inhibitor (C1-INH) concentrate ... highly sensitized patients. C1-INH is a human protein ... The study shows that post-transplant treatment ... levels of complement components 3 and 4, suggesting ...
(Date:7/30/2014)... July 30, 2014 While administrative ... health services research for a relatively long period ... interest regarding the use of these ‘big data’ ... , By combining today’s powerful computer technology with ... era, ‘big data’ holds significant promise for identifying ...
(Date:7/30/2014)... , July 30, 2014   Wyle ... the Fully Integrated Lifecycle Mission Support Services (FILMSS) ... Administration (NASA). Under the terms of the contract, ... Center at Moffett Field, Calif. In particular, Wyle ... bioscience flight development projects on the International Space ...
(Date:7/30/2014)... BCC Research ( http://www.bccresearch.com ) reveals in ... Industrial Applications , the global market for industrial enzymes ... 2018, registering a five-year compound annual growth rate (CAGR) ... in the detergent enzyme segment with a CAGR of ... influenced almost every sector of industrial activity, ranging from ...
Breaking Biology Technology:Study Suggests C1-INH May Aid in Prevention of Antibody-Mediated Rejection Following Kidney Transplant 2Study Suggests C1-INH May Aid in Prevention of Antibody-Mediated Rejection Following Kidney Transplant 3Leveraging ‘Big Data’ - Methodological Considerations in Health Services Research, New Webinar Hosted by Xtalks 2Wyle Wins NASA's Fully Integrated Lifecycle Mission Support Services Contract 2Global Market for Industrial Enzymes to Reach Nearly $7.1 Billion by 2018; Detergent Enzyme Market to Record Maximum Growth 2Global Market for Industrial Enzymes to Reach Nearly $7.1 Billion by 2018; Detergent Enzyme Market to Record Maximum Growth 3
... 1 million to 600 billion pages-that's one measure of ... years. , ,From the beginning, the Internet Scout Project ... finding, filtering and presenting online information. Internet Scout is ... also looking forward to a future of continuing exploration ...
... - Bruker AXS Inc. announced an agreement ... revenues of $5-6 million, from Princeton Gamma-Tech Instruments, Inc ... the acquisition is expected to close in the fourth quarter ... that an agreement had been reached to acquire Roentec ...
... Madison, Wis. A free online service, ... potential board members is up to 500 registered users, ... this year. , , Michael Best & Friedrich, LLP ... Solutions, LLC , began the service to connect organizations ...
Cached Biology Technology:Internet Scout celebrates 10 years on web, eyes new project launch 2
(Date:7/30/2014)... the first high-resolution map of the carbon stocks ... Per. The new and improved methodology used to ... future market-based carbon economies. The new carbon ... and it provides the critical input to studies ... use, and enforcement purposes. The technique includes the ...
(Date:7/29/2014)... As the climate warms and sea ice retreats, the ... season of increasingly open water which is predicted to ... of this century. Storms thus have the potential to ... a new and unpredictable element to the region. , A ... waves in the middle of the Arctic Ocean, and ...
(Date:7/29/2014)... microorganisms, such as bacteria and spermatozoa, are subjected to ... exerted by the surrounding fluid. Such low-level inertia ... that the direction of propulsion made possible by such ... fluid. These findings have been published in EPJ ... and Atomic Energy Commission (CEA), in Le Barp, France, ...
Breaking Biology News(10 mins):Peru's carbon quantified: Economic and conservation boon 2Peru's carbon quantified: Economic and conservation boon 3Huge waves measured for first time in Arctic Ocean 2Huge waves measured for first time in Arctic Ocean 3Optimum inertial self-propulsion design for snowman-like nanorobot 2
... of Pediatrics National Children,s Art Contest drew hundreds of entries ... the theme of "My Healthy Lifestyle," the 2011 contest invited ... exercise, sleep and limiting screen time. This was the Academy,s ... three age groups each receive a $500 cash prize ...
... has thought a lot about the challenges of power ... at Newark College of Engineering in 1960. He,s built ... technologies needed to produce the electricity that sustains virtually ... recipient of one of six NJIT Alumni Achievement Awards. ...
... of an organically grown apple may hold within it a ... of Washington have teamed up on a startup that promises ... crumbly charcoal-like product for farmers that helps their soil hold ... often improves yield for farmers. "Wine growers, organic farmers and ...
Cached Biology News:NJIT alum, power expert, advisor during Exxon/Valdez clean-up, gets award 2NJIT alum, power expert, advisor during Exxon/Valdez clean-up, gets award 3Turning slash piles into soil benefit 2
...
... benchtop fume hood ventilates and ... indoor release of exhaust. Bonded ... contaminants. Configurations are available with ... include final HEPA filter, work ...
...
... Staccato Series of systems provide fast and ... , genomics, proteomics and drug development laboratories. ... configurations; Mini Workstation Series, Application Series and ... Station is a remarkably compact system for ...
Biology Products: