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:9/17/2014)... Shimadzu Scientific Instruments introduces ... which offers a powerful separation method for ... provides a new level of knowledge and ... to scientists analyzing pharmaceutical impurities, proteins, lipids, ... Comprehensive 2D-LC combines two independent separation modes ...
(Date:9/17/2014)... PA & Rockville, MD (PRWEB) September 17, 2014 ... won a Small Business Innovation Research (SBIR) grant to ... immunotherapy. The award will provide ITI with approximately $255,000 ... Tree nuts, a leading cause of food allergy, affect ... cause of anaphylaxis behind peanut allergy. Tree nut exposure ...
(Date:9/17/2014)... , Sept. 17, 2014 /PRNewswire/ - SQI Diagnostics ... that develops and commercializes proprietary technologies and products for ... have been approved for trading in the ... Trading is expected to commence on September 17, 2014 ... will continue to trade on the TSX Venture Exchange ...
(Date:9/17/2014)... Escape Alert, LLC . filed international patents ... alert owners if their pet has escaped and enable them to ... in the pet’s body. This is the first implantable microchip ... been around for many years, they are passive until read by ... a pet’s location or alert the owner that the pet has ...
Breaking Biology Technology:Shimadzu’s New Nexera-e Two Dimensional Comprehensive Liquid Chromatograph Empowers Laboratories to Separate Even the Most Complex Mixtures 2Immunomic Therapeutics Awarded SBIR Grant to Develop Multi-Nut Allergy Immunotherapy 2Immunomic Therapeutics Awarded SBIR Grant to Develop Multi-Nut Allergy Immunotherapy 3SQI Diagnostics to begin Trading on the OTCQX Marketplace in the United States 2SQI Diagnostics to begin Trading on the OTCQX Marketplace in the United States 3Implantable Microchip for Pets with GPS Tracking - Patent Filed by Escape Alert, LLC. 2Implantable Microchip for Pets with GPS Tracking - Patent Filed by Escape Alert, LLC. 3
... train workforce, stimulate economy ALEXANDRIA, Va., May 7 ... budget request, proposing level funding for the Carl D. ... $1.3 billion. The Perkins Act is the primary source ... connect education and real-world careers. The Association for Career ...
... May 7 Elsevier,one of the world,s leading ... and services, announced today that Michael Hansen,CEO of ... in,light of recent allegations of improper Australia based ... "Elsevier prides itself on ...
... 7 The Sixth Annual Healthcare Unbound Conference & ... will feature the coming together of consumer and healthcare ... The Conference will take place at the Seattle ... Attendees will include a diverse representation of the ...
Cached Biology Technology:Obama Budget Level Funds Career Technical Education 2Obama Budget Level Funds Career Technical Education 3Statement From Michael Hansen, CEO of Elsevier's Health Sciences Division, Regarding Australia Based Sponsored Journal Practices Between 2000 and 2005 2Sixth Annual Healthcare Unbound Conference to Focus on Remote Monitoring, Home Telehealth & eHealth for Chronic Care Management & Wellness Promotion 2Sixth Annual Healthcare Unbound Conference to Focus on Remote Monitoring, Home Telehealth & eHealth for Chronic Care Management & Wellness Promotion 3
(Date:9/17/2014)... crime unit designed to fight food fraud in ... being passed off as beef scientists from Germany ... describe their approach, which represents a vast improvement over ... Food Chemistry . , Hans-Ulrich Humpf and colleagues note ... But they also say that adding, for example, horse ...
(Date:9/16/2014)... of severe maternal morbidity amongst women in Australia ... new study published today (17 September) in ... . , Australians generally enjoy high standards of ... disparities exist, in particular between indigenous and non ... explore the independent impact of socioeconomic position on ...
(Date:9/16/2014)... The 36 Pit Fire began on September 13, 2014. ... It is currently over 2,300 acres in size and ... The 36 Pit Fire was calm Monday night due to ... establishing an anchor point and beginning to construct a fireline ... The following resources are working on the fire: , 7 ...
Breaking Biology News(10 mins):New study examines the impact of socioeconomic position & maternal morbidity in Australia 2New study examines the impact of socioeconomic position & maternal morbidity in Australia 3
... at levels consumed by breast cancer patients and survivors in ... rats, new research found., The role of folate, a ... development and progression of breast cancer is highly controversial. ... breast cancer, recent studies have suggested that taking high amounts ...
... When the temperature rises on Baffin Island, in the ... ice for thousands of years, are exposed. Using radiocarbon ... has calculated the age of relic moss samples that ... moss samples would have been destroyed by erosion had ...
... the Montreal Neurological Institute and Hospital in Canada ... Parkinson,s disease are involved in the early-stage quality ... reported in T he EMBO Journal , removes ... mitochondria., "PINK1 and parkin, are implicated in selectively ...
Cached Biology News:Large amounts of folic acid shown to promote growth of breast cancer in rats 2Quality control of mitochondria as a defense against disease 2
Mouse monoclonal [6G11] to Cytohesin 1 + 2 ( Abpromise for all tested applications)....
HNF-1 Purified Anti-Mouse, Anti-Rat, Anti-Rabbit, Anti-Human clone 2, Isotype Mouse IgG 1 , 50 µg Consult technical datasheet for details....
Recombinant Porcine IL-1 alpha/IL-1F1, CF...
... siSolutions siRNA Custom Synthesis ... effective duplex RNA to knockdown ... 21-mers (19 RNA base pairs ... recently-published 27-mers, or other lengths ...
Biology Products: