Navigation Links
High-Efficiency Site-Directed Mutagenesis of Large Plasmid Vectors

n with an error rate that is significantly lower than conventional thermostable enzymes5 minimizing undesirable second-site mutations.

Improving Mutagenesis of Large Plasmids

In the new QuikChange XL kit, modifications have been made to the QuikChange method to improve the efficiency of mutagenizing large or difficult plasmid vectors. In addition, XL10-Gold ultracompetent cells have been included to ensure the highest transformation efficiencies possible (Figure 1). The transformation efficiency of XL10-Gold cells is 5-fold higher than the transformation efficiency of XL1-Blue cells employed in the standard QuikChange kit6. Moreover, XL10-Gold cells contain the Hte phenotype, which increases transformation efficiency of larger DNA plasmids.

The performance of the QuikChange XL kit is demonstrated in mutagenesis studies employing a 10.1-kb plasmid. In this study, primers were designed to repair a frameshift mutation by inserting the missing nucleotide in the open reading frame (ORF) of the Moloney Murine Leukemia Virus (MMLV) gag-pol fusion protein. QuikChange reactions were performed with components of the standard kit or the QuikChange XL kit. Electrophoresis of Dpn I-digested reaction products showed that bands of the appropriate size (10.1-kb) were only evident in reactions performed with components of the QuikChange XL kit (Figure 2, lane 1). Moreover, transformation of XL10-Gold cells with a QuikChange XL reaction produced 32 colonies. In contrast, no colonies were produced with the standard QuikChange kit (Table 1). Nine out of 10 colonies produced a plasmid with the correct base insertion, as evidenced by DNA sequence analysis, and restoration of gag-pol gene function was verified by assaying reverse transcriptase
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. High-Efficiency Transfections Achieved with New Low-Toxicity Reagent
2. Big Blue Rodent Cell Cultures for Mutagenesis Studies
3. Randomize Gene Sequences with New PCR Mutagenesis Kit
4. PCR-Grade Nucleotides Larger Pack Sizes Now Available
5. Rapidly Synthesize Large Amounts of Capped RNA
6. Efficient Cloning and Electro-transformation of Large Eukaryotic DNA Fragments
7. Sub-Micron Gold Particles Are Superior to Larger Particles for Efficient Biolistic Transformation of Organelles and Some Cells
8. Efficiently Insert Unique Restriction Sites into Plasmid Vectors
9. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
10. Generate High-Quality, Directional Plasmid cDNA Libraries
11. New Reporter Plasmids for Studying Interferon-Stimulated Signal Transduction Pathways
Post Your Comments:
(Date:8/22/2014)... Aug. 22, 2014 /PRNewswire/ - BIOREM Inc. (TSXV: BRM) ("Biorem" or "the ... ended June 30, 2014.  Biorem,s complete 2014 second quarter financial statements and ... Summary:Three-months ended June 30, , Six-months ended June ... , 2013 , 2014 , 2013 ... , 9,195 Gross profit , 56 , ...
(Date:8/22/2014)... published today (August 22nd), in the scientific journal ... Neuroscience Programme establish the effect of serotonin on ... genetic and optical techniques., "Serotonin is a small ... range of brain functions, from the control of ... emotional behaviours, This neurotransmitter is also popularly thought ...
(Date:8/22/2014)... investigated for application as catalysts, sensors, drug ... in photonics and molecular electronics. Gaining knowledge ... physical and chemical properties, has been challenging. ... demonstrated that high-resolution electron microscopy can be ... which all gold atoms are observed. The ...
(Date:8/22/2014)... The Global and China Diabetes Drugs Industry ... state of the Diabetes Drug industry in Global and ... the industry, including definitions, applications and industry chain structure. ... provided with a focus on history, developments, trends and ... international and Chinese situation is also offered. , The ...
Breaking Biology Technology:Biorem Reports 2014 Second Quarter Results 2Biorem Reports 2014 Second Quarter Results 3From happiness to pain: Understanding serotonin's function 2A breakthrough in imaging gold nanoparticles to atomic resolution by electron microscopy 2Diabetes Drugs Industry Growth Analysis in Global, China Regions Research Report Available at DeepResearchReports.com 2Diabetes Drugs Industry Growth Analysis in Global, China Regions Research Report Available at DeepResearchReports.com 3
... , MISSOULA, Mont., June 24 VEGA PROMOTIONAL SYSTEMS, ... owned subsidiary, Natural Fuels Industries, it is planning to ... to produce alternative energy from organic waste. , ... "America,s Best Places For Alternative Energy," the abundance of ...
... BOTHELL, WA, and VANCOUVER, June 24 /PRNewswire-FirstCall/ - OncoGenex ... that the company has reached an agreement with the ... protocol assessment process (SPA) on an amendment to the ... its lead product candidate targeting castrate resistant prostate cancer ...
... Higher Percentage of Patients Have Preventative Testing , ... Advice Networks , the nation,s leading provider of ... Healthy Advice educational programs, 16.9 percent more ... percent more patients had colonoscopies (colon cancer screening) compared ...
Cached Biology Technology:Natural Fuels Industries Eyes 'Biomass Rich' Georgia for Production Facility 2Natural Fuels Industries Eyes 'Biomass Rich' Georgia for Production Facility 3OncoGenex Pharmaceuticals Completes Amendment to Phase 3 Special Protocol Assessment for Confirming Survival Benefit in Patients Receiving OGX-011 with First-Line Docetaxel for Metastatic Prostate Cancer 2OncoGenex Pharmaceuticals Completes Amendment to Phase 3 Special Protocol Assessment for Confirming Survival Benefit in Patients Receiving OGX-011 with First-Line Docetaxel for Metastatic Prostate Cancer 3OncoGenex Pharmaceuticals Completes Amendment to Phase 3 Special Protocol Assessment for Confirming Survival Benefit in Patients Receiving OGX-011 with First-Line Docetaxel for Metastatic Prostate Cancer 4OncoGenex Pharmaceuticals Completes Amendment to Phase 3 Special Protocol Assessment for Confirming Survival Benefit in Patients Receiving OGX-011 with First-Line Docetaxel for Metastatic Prostate Cancer 5Healthy Advice Networks Announces Improved Patient Outcomes in Physicians' Offices with Healthy Advice Programs 2
(Date:8/21/2014)... previously unknown process for harvesting energy and producing oxygen ... State University scientist has discovered. The discovery lays the ... harvesting energy from the Sun, and understanding dense blooms ... lakes worldwide. A paper describing the discovery will be ... the journal Science on 21 August 2014. ...
(Date:8/21/2014)... Israel Deaconess Medical Center (BIDMC ) are among the ... Minds 2014," a comprehensive list compiled by ... science metrics and research performance analysis. , ... performing and publishing work that their peers recognized as ... a Thomson Reuters statement. Researchers were identified based on ...
(Date:8/21/2014)... study recently published in the journal Carcinogenesis ... new role for the protein adenomatous polyposis coli (APC) ... cancer-related deaths in the U.S. , Lead author Kristi ... and co-leader of the Cancer Biology program at the ... her career trying to understand the various activities of ...
Breaking Biology News(10 mins):Hot-spring bacteria reveal ability to use far-red light for photosynthesis 2Hot-spring bacteria reveal ability to use far-red light for photosynthesis 3BIDMC researchers named among 'the most influential scientific minds' 2BIDMC researchers named among 'the most influential scientific minds' 3Research offers insight into cellular biology of colorectal cancer 2Research offers insight into cellular biology of colorectal cancer 3
... The citrus disease Huanglongbing (HLB), meaning "yellow shoot ... in English-speaking countries, is the most destructive disease ... tools and management strategies are desired to control ... phosphorus deficiency as a contributing factor in symptom ...
... species of scorpion, Vaejovis brysoni, was found in the ... the same group also inhabits this mountain range, making this ... species distributed on the same mountain. The study was published ... . These mountains overlook the city of Tucson, Arizona. ...
... Sloan Foundation is pleased to announce the selection of ... Sloan Research Fellowships for 2013. , Awarded annually ... and scholars whose achievements and potential identify them as ... "The Sloan Research Fellows are the best of the ...
Cached Biology News:Phosphorus starvation linked to symptoms of citrus disease Huanglongbing in new study 2Phosphorus starvation linked to symptoms of citrus disease Huanglongbing in new study 3Sloan Foundation announces 2013 Sloan Research Fellows 2
Contains 100 positive controls for effective screening of HDAC inhibitors by fluorometric method....
dynactin 4, mRNA (cDNA clone MGC:3248 IMAGE:3505855), complete cds...
One-step, microplate or cuvet, colorimetric, detection range 0.01 g/dL to 5 g/dL. Procedure: 5 min....
MOUSE ANTI PROTEIN KINASE C BETA 2 Immunogen: Protein kinase C beta 2...
Biology Products: