Navigation Links
Mammalian Expression Vector for Efficient Cloning of PCR Fragments

t PCR cloning kit was verified using a test gene. To reduce the number of potential mutations that result during PCR amplification and to generate blunt-ended DNA fragments, we chose Pfu DNA polymerase for PCR amplification of the ampicillin-resistance gene. The pCMV-Script vector was digested with Srf I restriction endonuclease and ligated to the PCR-amplified ampicillin-resistance gene in the presence of Srf I to eliminate the background of recircularized vector. This mixture was transformed into XL10-Gold cells, and 50 l of the 500 l transformation mix was plated on LB agar plates containing kanamycin. In order to determine the percentage of colonies that contained the insert, 100 of the resulting 250 colonies were streaked onto an LB agar plate with ampicillin. Of the recovered transformants, 73% contained the ampicillin-resistance-gene insert.

Testing Mammalian Expression

The pCMV-Script vector was tested in both transient and stable transfection assays. The green fluorescent protein gene (GFP) and the firefly luciferase gene were used for transient transfection assays. The GFP gene was cloned into the pCMV-Script vector, and the resulting construct was transfected using standard lipid transfection methods into Chinese hamster ovary (CHO) cells. The expression of the GFP gene product was detected by Western blot analysis (data not shown).

Figure 2

Similarly, the luciferase gene was cloned into the pCMV-Script vector and used to transfect CHO cells. In order to compare expression levels of genes inserted into the pCMV-Script vector with expression levels in other available vector products, the luciferase gene was also inserted into the pcDNA 3.1 vector. Each construct and appropriate controls were used to transfect CHO cells. Forty-eight hours following transfection,
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. New Mammalian Two-Hybrid System Detects Protein-Protein Interactions
2. Highest Transfection Efficiency of an Endotoxin-Sensitive Mammalian Cell Line
3. An Epitope Tagging Vector for Gene Expression in Mammalian Cells
4. Versatile Vectors for Ponasterone A- Inducible Control of Gene Expression in Mammalian Cells
5. New Mammalian Expression Vectors Employ Stable, High-Level Fluorescence Humanized Renilla GFP Reporter
6. A New Lambda Vector for Mammalian Expression
7. Mammalian and Bacterial Expression in One Vector
8. Mammalian Expression Vectors with Interchangeable Selectable Markers
9. Low-Toxicity, Lipid-Mediated Transfection of Mammalian Cells
10. Mycoplasma Contamination Reduces the Effect of Lipid-Mediated Transfection of Mammalian Cells
11. Reagents for Up and Down-Regulation of miRNA Activity in Mammalian Cells
Post Your Comments:
(Date:8/31/2015)... Tampa, FL (PRWEB) , ... August 31, 2015 ... ... will bring together the world's most successful food and beverage processors, equipment manufacturers ... its competencies in product inspection and industrial weighing and formulation for manufacturers of ...
(Date:8/28/2015)... , August 28, 2015 According ... (Media, Sera, Cryogenic Storage Systems, Thawing Equipment, Alarms), by ... Medicine, Clinical Trials), End User - Global Forecast to ... expected to reach USD 3,731.03 Million by 2020 from ... of 11.65% between 2015 and 2020. ...
(Date:8/28/2015)... (PRWEB) , ... August 28, 2015 , ... Since the initial launch of FireflySci Inc. ... quirky humor and excellent customer service. Images such as the Fonz in a lab ... add some flavor to the humdrum spectroscopy field. , FireflySci is proud to ...
(Date:8/27/2015)... Aug. 27, 2015 The National Necrotizing ... eyes—and life—were saved by doctors at Vanderbilt University Hospital ... NNFF, NovaBay Pharmaceuticals and Dr. John Crew , ... at Seton Medical Center in Daly City, ... a breakthrough approach, pioneered by Dr. Crew, that had ...
Breaking Biology Technology:METTLER TOLEDO Hosts Food Safety Educational Center at 2015 PROCESS EXPO 2Biopreservation Market Worth 3,731.03 Million USD by 2020 2Biopreservation Market Worth 3,731.03 Million USD by 2020 3Biopreservation Market Worth 3,731.03 Million USD by 2020 4Biopreservation Market Worth 3,731.03 Million USD by 2020 5Cuvette Manufacturer FireflySci Launches Back to School Deals with Complimentary Shipping On All Orders 2Patient's Life and Eyes Saved from Gruesome Flesh-Eating Infection Consuming Her Face 2Patient's Life and Eyes Saved from Gruesome Flesh-Eating Infection Consuming Her Face 3
... Pa., Dec. 24 Wyeth,Pharmaceuticals, a division of ... will,pursue a patent infringement claim for lost profits ... from Teva,s launch of a,pantoprazole tablet -- a ... not disputed that its product infringes the United ...
... presents a new marvel of just how much memory can ... nanolasers being developed in Sakhrat Khizroevs lab at the University ... smaller. , As reported in the latest issue of ... tiny that they point to a future where a 10-terabit ...
... WALTHAM, Mass., Dec. 21 Inverness Medical,Innovations, Inc. ... marketer of rapid,diagnostic products for the consumer and ... upcoming JP Morgan Healthcare Conference,being held January 7 ... Hotel in,San Francisco. Ron Zwanziger, Chief Executive Officer, ...
Cached Biology Technology:Wyeth Will Pursue Claim for Damages Resulting From Teva Pharmaceuticals' Launch of Generic Pantoprazole in Violation of PROTONIX Patent 2Wyeth Will Pursue Claim for Damages Resulting From Teva Pharmaceuticals' Launch of Generic Pantoprazole in Violation of PROTONIX Patent 3Wyeth Will Pursue Claim for Damages Resulting From Teva Pharmaceuticals' Launch of Generic Pantoprazole in Violation of PROTONIX Patent 4The Library of Congress in your wrist watch? 2Inverness Medical Innovations to Participate at JP Morgan Healthcare Conference on January 8, 2008 2
(Date:8/26/2015)... , August 26, 2015 The ... and Five Factor), Application (Travel & Immigration, Government, Banking, ... Global Forecast to 2020", published by MarketsandMarkets, Multi-Factor Authentication ... 2020, growing at a CAGR of 17.7% between 2015 ... 82 F igures spread through 169 ...
(Date:8/20/2015)... , Aug. 20, 2015   Sensory, ... on improving the user experience and security of ... technologies, today announced that its TrulySecure™ ... authentication software to be FIDO Certified™. ... for compliance with the FIDO UAF (Universal Authentication ...
(Date:8/17/2015)... , Aug. 17, 2015  NXT-ID, Inc. (NASDAQ: ... company focused on the growing mobile commerce market and ... sales campaign for Q4 2015. The new marketing campaign ... consisting of Amplitude Marketing Group ( http://amplitudemarketing.com/ ) and ... is a leader in retail driven marketing and brand ...
Breaking Biology News(10 mins):Multi-Factor Authentication Market Worth 9.60 Billion USD by 2020 2Multi-Factor Authentication Market Worth 9.60 Billion USD by 2020 3Multi-Factor Authentication Market Worth 9.60 Billion USD by 2020 4Multi-Factor Authentication Market Worth 9.60 Billion USD by 2020 5TrulySecure From Sensory Becomes First Face and Voice Biometrics Technology to be FIDO UAF Certified 2TrulySecure From Sensory Becomes First Face and Voice Biometrics Technology to be FIDO UAF Certified 3TrulySecure From Sensory Becomes First Face and Voice Biometrics Technology to be FIDO UAF Certified 4TrulySecure From Sensory Becomes First Face and Voice Biometrics Technology to be FIDO UAF Certified 5NXT-ID, Inc. Launches Sales Campaign for Wocket Smart Wallet with Award Winning Marketing Team 2NXT-ID, Inc. Launches Sales Campaign for Wocket Smart Wallet with Award Winning Marketing Team 3
... Boston, MA Using 120 years of feathers ... University researchers have been able to track increases in ... nigripes ), an endangered seabird that forages extensively throughout ... the observed increase in methylmercury levels, most likely from ...
... University of the Negev software engineering students have developed ... computer without using a keyboard or mouse only ... to develop devices to read brainwaves and operate specific ... a laboratory setting. The BGU ...
... April 18, 2011 Mention vitamin D-fortified foods and ... with the sunshine vitamin since the 1930s. A new ... Challenges/Chemistry Solutions" podcast series says that vitamin D-fortified bread ... essential nutrient that is difficult to get naturally in ...
Cached Biology News:Mercury on the rise in endangered Pacific seabirds 2
Shake 'n' Stack oven - New Model (includes 10 bottle capacity rotisserie, adjustable feet, drip tray, manual)...
... Cell Expansion Kit - Monolayer Plus is a ... expansion as a monolayer in a serum-free environment. ... Media Supplement, which has been optimized for neural ... . (1996) Genes & Development 10 :3129). ...
... thymus Description: Topoisomerase I relaxes both ... breakage and rejoining of phosphodiester bonds (1,2) . ... to be differentiated by a single nucleotide pair ... active in the absence of Mn 2+ ...
... catalyzes the template-directed polymerization of nucleotides into ... Polymerase I possesses a 3?5 exonuclease activity ... rate during DNA replication, and also contains ... enzyme to replace nucleotides in the growing ...
Biology Products: