Navigation Links
Efficient DNA transfection of primary CNS neurons using TransMessenger ,,, Transfection Reagent

on poly-D-lysine-coated 96-well plates at a density of 120,000 cells/well in a volume of 200 l/well.

Cells were transfected in a 96-well format on the seventh day after plating. The indicated amounts of DNA (0.1-0.5 g/well of a GFP- or -gal-encoding plasmid) were condensed for 5 minutes with Enhancer R and Buffer EC-R in a total volume of 30 l. The ratio of DNA to Enhancer R was 1:4. TransMessenger Reagent (1-4 l) was added in a total volume of 20 l Buffer EC-R and complex formation proceeded for 10 minutes. 100 l Neurobasal/B27 medium was added to the transfection complexes, medium was removed from the cells, and the complexes were transferred to the cells. After 6 hours of incubation, the complexes were removed and fresh medium was added.

For immunofluorescence, cells were fixed using 4% paraformaldehyde 24 hours after transfection. Immunofluorescence was performed according to standard procedures using primary antibodies directed against MAP2 (a neuron-specific marker protein) and GFAP (an astrocyte-specific marker protein) and Cy3-labeled secondary antibodies.

-galactosidase activity was measured 48 hours after transfection. Cells were lysed and enzyme activity was quantified by a colorimetric assay using ONPG and a -galactosidase standard curve.

Results and discussion

To optimize the transfection protocol, transfections were performed using different amounts of plasmid DNA and TransMessenger Reagent. In each well of a 96-well plate, 0.1, 0.3, or 0.5 g plasmid DNA were used, in combination with 1-4 l of TransMessenger Reagent. Six replicates were set up for each combination.

Transfection efficiencies were quantified by measurin
'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. Efficiently Insert Unique Restriction Sites into Plasmid Vectors
2. Efficient Cleavage of Fusion Proteins to Yield Native Amino Termini
3. Mammalian Expression Vector for Efficient Cloning of PCR Fragments
4. Efficient Transfection of Neurospora Crassa
5. Efficient Transfection of Neurospora Crassa
6. Efficient Recovery of Ultrapure Plasmid DNA
7. Efficient and Reliable PCR Setup Using Eppendorf MasterMix
8. Efficient RNAi-mediated gene silencing in neuronal cells using QIAGEN siRNA and TransMessenger Transfection Reagent*
9. Cloning Based on Efficient Three-Fragment Assembly DNA Ligation
10. Efficient and Reliable Linear Amplification of cRNA
11. Efficient Delivery of siRNAs to Human Primary Cells: Electroporation vs. Chemical Transfection
Post Your Comments:
(Date:9/18/2014)... KEEGO HARBOR, Mich. , Sept. 18, 2014  Health ... -based biotech firm investigating early-stage algae bioactive compounds and metabolic ... member to the company,s board of directors. Ms. Nola ... executive and investment advisor joined the board effective September 17, ... have Ms. Masterson agree to join our board," states ...
(Date:9/18/2014)... 18, 2014 Texas Governor Rick ... Texas A&M Health Science Center CEO Brett Giroir, ... Health and Human Services (HHS), State of Texas ... pandemic influenza vaccine manufacturing facility in Bryan, Texas, ... for the Texas A&M Biocorridor – a rapidly ...
(Date:9/18/2014)... Sept. 18, 2014 Research and Markets ... and Chinese Stem Cell Industry Report, 2014-2017" report ... undifferentiated biological cells that can differentiate into specialized cells ... cells. Stem cell therapy can be applied to treatment ... disease), nervous system diseases, damage or lesion of liver, ...
(Date:9/18/2014)... Research and Markets has announced the addition ... report to their offering. The ... and in-depth study on the current state of the ... a basic overview of the industry including definitions, classifications, ... analysis is provided for the international markets including development ...
Breaking Biology Technology:Health Enhancement Announces New Board Member In Advance Of Annual Shareholder Meeting 2Texas A&M Dedicates National Pandemic Influenza Vaccine Manufacturing Facility, on Track for 2016 Start-Up Phase 2Texas A&M Dedicates National Pandemic Influenza Vaccine Manufacturing Facility, on Track for 2016 Start-Up Phase 3Texas A&M Dedicates National Pandemic Influenza Vaccine Manufacturing Facility, on Track for 2016 Start-Up Phase 4Global and Chinese Stem Cell Industry Report, 2014-2017 2Global and Chinese Stem Cell Industry Report, 2014-2017 3Global Human Albumin Industry Report 2014 2Global Human Albumin Industry Report 2014 3
... Sheboygan, Wis. - Gov. Jim Doyle ... the Wisconsin Aerospace Authority - both of whom expressed ... for future space travel. , ,Mark Hanna, a city ... program, will join several already-named nominees for the commission, ...
... will have the exclusive global distribution rights to a stem ... with its developer, ThermoGenesis . , ,Under the terms ... product, known as the Auto Express System, to the ... of private umbilical cord blood banks. The AXP System extracts ...
... took another turn last month when the District Court of ... Cigar, Inc ., v. GoTo.com, Inc . , ,Plaintiff ... of Yahoo! , ,GoTo sold several keywords, ... various JR Cigar competitors. , ,As mentioned in my prior ...
Cached Biology Technology:Wisconsin spaceport committee moves closer to formation 2Are consumers confused by search engine ads? 2Are consumers confused by search engine ads? 3Are consumers confused by search engine ads? 4
(Date:9/18/2014)... tiny strands of nucleic acid that contain instructions for ... data, our DNA is copied into RNA molecules, which ... in our cells. , Several years ago, scientists ... other known RNAs, this molecule is circular, and was ... has been known about how they are produced. Moreover, ...
(Date:9/18/2014)... and ethnic groups describe losing eyesight as potentially ... more so than other conditions including: loss of ... 49% of non-Hispanic whites, 43% of Asians and ... ailment is the worst that could happen to ... AIDS/HIV. Hispanics and Asians ranked cancer first ...
(Date:9/18/2014)... devastated algal forests in the eastern Mediterranean Sea pose ... their distribution continues to expand as the climate warms, ... team of researchers led by Dr Adriana Vergs of ... Institute for Advanced Studies in Spain, is published in ... team surveyed more than 1000 kilometres of coastline in ...
Breaking Biology News(10 mins):Scientists find how mysterious 'circular RNA' is formed, claim muscular dystrophy link 2Americans rate losing eyesight as having greatest impact on their lives 2Americans rate losing eyesight as having greatest impact on their lives 3Tropical fish a threat to Mediterranean Sea ecosystems 2
... in protecting us from diseases, but how does it ... that before dendritic cells move to the lymph nodes ... move much faster. Immature dendritic cells patrol the tissues ... they undergo a rigorous maturation process. During this maturation ...
... A stunning discovery based on epigenetics (the inheritance of ... nutrient found in eggs and other foodsduring pregnancy may ... This finding by a team of biologists at Boston ... pregnancy to breast cancer. It also is the first ...
... who have had the common chest wall deformity known ... ability to exercise, according to a study published December ... conducted at 11 North American hospitals, involved telephone interviews ... 8 and 21 who had pectus excavatum surgery. ...
Cached Biology News:Immune cells reveal fancy footwork 2Eating eggs when pregnant affects breast cancer in offspring 2Marked improvement in body image, physical stamina, post-surgical pectus patients report 2Marked improvement in body image, physical stamina, post-surgical pectus patients report 3
... applications for the use of small interfering ... via the RNA interference (RNAi) pathway have ... molecular biology fields. siRNA for experimental use ... or enzymatically, and then transfected into the ...
... is commonly used as a fusion partner ... The GSTTag sequence has been reported to ... the solubility of its fusion partners. When ... GSTTag fusion proteins can be purified with ...
a1-Antitrypsin (human, alpha-1 AT)...
... demands of the routine trace metal analytical ... robust and simple to use instrumentation. The ... most challenging sample matrices found in the ... the toughest analytical challenges found in the ...
Biology Products: