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The researchers aimed to bioengineer a new red fluorescent protein tag from cyanobacteriochromes in Thermosynechococcus elongatus. They transfected Jurkat cells with plasmids containing fluorescent protein tags for visualization under advanced microscopes. Electroporation and the plasmid pAcGFP1-Tubulin were found to be most efficient for transfection. Future goals include optimizing expression of their engineered 569 fluorescent tag in mammalian cells.
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The document discusses engineering a red fluorescent protein tag from the cyanobacterium Thermosynechococcuselongatus for use as an easily detectable protein marker. The tag would respond to different light wavelengths and could be used to study protein expression and transformation in T. elongatus cells. Existing green fluorescent protein tags from jellyfish require a different light wavelength to be detected. The engineered red tag would use the GAF domain protein and phycocyanobilin chromophore from T. elongatus to fluorescently label and image proteins when expressed in host cells.
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- Expression of the tRNA intron splicing endonuclease protein was induced in E. coli and detected via western blotting.
- The expressed protein was purified using affinity chromatography for future structural and functional studies.
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1) TAPS provides high conversion rates of 5mC to thymine without affecting unmodified cytosines or damaging DNA.
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Real-time PCR primer
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Microscope
This document provides an introduction and overview of microscopes. It discusses the basic parts of a microscope including the eyepiece, objective lenses, stage, and condenser lens. It explains how to properly use and focus a microscope. The document also covers the different types of microscopes, including compound, stereoscopic, electron, and specialized microscopes. It provides descriptions of optical and electron microscopes and their distinguishing features.
Lab protocols by Kojo Ahiakpa.
This document provides instructions for several microscopy lab activities:
1. It describes how to properly use a microscope, including how to prepare slides, focus the lens, and switch between low and high magnification.
2. Students observe onion cells under the microscope and identify cell structures like the nucleus and cell wall.
3. Methods are outlined for estimating the size of onion cells using the microscope's field of view.
4. Students look for stages of mitosis in root tip cells through staining and microscopy.
How to use a microscope
Microscopes allow observation of objects too small to be seen with the naked eye. They were instrumental in discovering that cells are the basic unit of life and identifying disease-causing microorganisms. A microscope has a stage to hold slides, a light source, lenses that refract light to magnify the specimen, objective lenses that can be changed for different magnifications, and eyepieces. Specimens must be thin to view under the microscope and are placed on slides with a cover slip. Temporary slides can be made from pond water or cheek cells, while permanent slides include plant stems.
Parts Of The Microscope
The document describes the main parts of a microscope and their functions. It includes labels for the eye piece, objectives, stage, adjustments, light source, and other basic components. When using the microscope, slides are placed on the stage and the user looks through the eye piece while adjusting the objectives and fine adjustment to focus on the sample.
Parts of a microscope
The document describes the main parts of a microscope and their functions, including the eyepiece, body tube, nosepiece, objectives, arm, stage, stage clips, diaphragm, coarse and fine adjustment knobs, light source, and base. It explains that objectives provide magnification and that total magnification is calculated by multiplying the eyepiece and objective magnifications.
The Microscope
The document discusses the history and components of the microscope. It explains that the first compound microscope was created in the 1590s in Holland by Hans and Zacharias Janssen. Later, Anthony van Leeuwenhoek and Robert Hooke improved microscope design by working on lens techniques. The key parts of a microscope are described as the body tube, objective and ocular lenses, stage, light source, and adjustments for focusing. Magnification is calculated by multiplying the ocular and objective lens powers. Care and proper use of microscopes is also outlined.
Compound microscope
A compound microscope uses two or more convex lenses to magnify objects. It has an objective lens close to the sample that produces a real, inverted image, and an eyepiece lens that further magnifies this image for viewing. Compound microscopes typically have 4 objective lenses providing 4x, 10x, 40x, and 100x magnifications. The two lenses work together to compound the magnification, allowing much higher total magnifications than a simple microscope.
History of the Microscope
The microscope was invented in 1595 by Hans and Zacharias Janssen in Holland. It was later perfected in the 17th century by Robert Hooke in England and Anton van Leeuwenhoek in the Netherlands. Van Leeuwenhoek was the first to observe bacteria, yeast, and single-celled organisms using early compound microscopes. Industrialization in the 18th-19th centuries led to standardized microscope parts and mass production, increasing access and new discoveries. Electron microscopes developed in the 1930s allow for higher magnifications over light microscopes.
The microscope presentation
The microscope has evolved significantly since its earliest forms in the 1st century AD. Some key developments include:
1) The earliest microscopes in the 1st century AD had simple lenses that provided around 10x magnification.
2) In the late 16th century, experiments using multiple lenses in a tube led to improved magnification. Galileo later established the optical principles behind using lenses.
3) In the 17th century, Anton van Leeuwenhoek made tiny lenses that greatly increased magnification and allowed him to discover bacteria and microorganisms, while Robert Hooke used early microscopes to observe plant and animal cells.
Compound microscope
- The document discusses the compound microscope, its history, parts, functions, and use.
- A compound microscope has more than one lens and was invented in the 1590s in the Netherlands. It allows higher magnification than simple microscopes.
- The main parts are the mechanical stage, optical system with objectives and eyepieces, illumination system, and adjustment controls. Objectives magnify the specimen while eyepieces provide a final view.
- Focusing involves using coarse and fine adjustments to bring the specimen into view at different magnifications including with immersion oil.
Parts of the microscope and their functions (Game)
This document lists and describes the main parts of a microscope: the arm supports the tube and connects it to the base; the stage holds the slide containing the specimen; the mirror focuses the light. It also describes the body tube, coarse adjustment knob, fine adjustment knob, base, eyepiece lens, clip, nosepiece, objective lens, diaphragm, and condenser.
The Microscope
This presentation summarizes the use of both the compound and electron microscopes. It fits the Biology IGCSE syllabus.
Introduction To Microscopes History & Parts
The document provides an introduction and brief history of microscopes from simple glass magnifiers in the 1600s to modern compound microscopes. It describes some of the key developments including the first compound microscope in the 1600s, improvements in the 1700s and 1800s using brass and iron, and modern features. It then explains how microscopes work similarly to telescopes but with smaller objective lenses due to examining tiny close specimens. It identifies the main parts of a compound microscope and their functions.
tHE lIGHT mICROSCOPE
The document discusses different types of light microscopes, including their parts, advantages, and uses. A bright field microscope has a simple setup and allows viewing live cells but is useless for some living specimens. A dark field microscope provides high quality images of raised features and is used for live biological samples. A phase contrast microscope does not require staining and allows examination of living cells. A fluorescent microscope detects cell structures and molecules and is used for living cells stained with fluorescent dyes.
compound microscope (basic)
The document discusses compound light microscopes. It explains that compound microscopes use multiple lenses to magnify specimens and have different light sources - some use mirrors to focus sunlight while others use illuminators. It then describes the main parts of a compound microscope - the eyepiece, body tube, objectives, stage, arm, base, illuminator, nosepiece, condenser, and diaphragm. Finally, it discusses how light travels through the microscope and some common uses of compound microscopes, such as pathology, forensics, education, and biology.
20 Respiratory System
The respiratory system allows for gas exchange between the lungs and bloodstream. It consists of an upper respiratory tract including the nose and throat, and a lower respiratory tract including the trachea, bronchi and lungs. The lungs contain tiny air sacs called alveoli where oxygen and carbon dioxide are exchanged through the blood vessels. The respiratory system functions through breathing which involves inhaling air through the nose and mouth, and exhaling air out through the mouth.
Types of Microscope
Microscopes are instruments designed to produce magnified images of small objects. They must accomplish three tasks: produce a magnified image, separate details in the image, and render details visible. There are different types of microscopes including simple, compound, stereoscopic, electron, scanning electron, and transmission electron microscopes. Electron microscopes use a beam of electrons instead of light to magnify images and can achieve higher magnifications than light microscopes. Confocal laser scanning microscopes use a laser beam to generate 3D images of thick specimens.
Microscope types and use
This document provides an introduction to different types of microscopes. It discusses light microscopes which use lenses to magnify objects from 40x to 400x. Stereoscopes allow binocular viewing from 10x to 20x magnification and create a 3D view. Scanning electron microscopes use electrons instead of light to magnify up to 2 million times but cannot view living specimens. Transmission electron microscopes also use electrons passed through very thin specimens to see inside cells. Different illumination techniques make some specimen parts more distinct. Total magnification is calculated from the objective and eyepiece lenses. Online resources for virtual microscopes are also provided.
Parts and functions of a compound microscope
The document describes the main parts and functions of a compound microscope, including mechanical parts like the base and stage that support the microscope, magnifying parts like the objectives and ocular that enlarge specimens, and illuminating parts like the mirror and condenser that provide light. It also explains how to properly use a compound microscope, such as carrying and focusing it, as well as techniques for preparing slides including mounting, staining, and examining specimens.
Microscopy
The document discusses different types of microscopy techniques. It provides details on:
1) Compound light microscopes which can magnify objects up to 2000x and transmit light through lenses to produce enlarged images.
2) Bright field microscopy which uses visible light and staining to produce contrasting images at magnifications up to 1000x.
3) Dark field microscopy which illuminates specimens on a dark background without staining, allowing observation of unstained samples.
4) Phase contrast microscopy which converts phase shifts in light waves into brightness contrasts, enhancing detail in living cells without staining at magnifications up to 400x.
5) Scanning electron microscopes which use electron beams at 100,000x magnification to
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We developed a homogeneous luminogenic annexin V binding assay to detect apoptosis in real time using a multimode plate reader. The detection reagent has two different annexin V fusion proteins engineered to contain complementing domains of a binary luciferase, a substrate for luciferase and a cell impermeable fluorogenic DNA dye to detect necrotic cells. The method allow real-time monitoring of Cellular apoptosis and necrosis in microwell plates without washing steps with a highly sensitive luminescent signal. The AnnexinV luminescent method is amenable to High throughput and is a good alternative to FACS, low-throughput Annexin V-FITC based method.
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The document summarizes research on expressing HIV-1 antigens in plants as a potential vaccine. The researchers found that transient expression in plants is a viable alternative to transgenic plants. They determined that targeting the HIV-1 Gag protein Pr55Gag and its derivative p17/24 to chloroplasts yielded the highest levels of these proteins. Mice vaccinated with p17/24 produced from chloroplasts developed strong CD4+ and CD8+ T cell responses and HIV-specific antibodies, showing it is a promising vaccine candidate.
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The document summarizes research on expressing HIV-1 antigens in plants as a potential vaccine. The researchers found that transient expression in plants is a viable alternative to transgenic plants. They determined that targeting the HIV-1 Gag protein Pr55Gag and its derivative p17/24 to chloroplasts yielded the highest levels of these proteins. Mice vaccinated with p17/24 produced from chloroplasts developed strong CD4+ and CD8+ T cell responses and HIV-specific antibodies, showing it is a promising vaccine candidate.
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1. Tomato production is affected by various bacterial, fungal and viral diseases which can cause considerable yield losses.
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This document provides the timetable and protocols for a practical course on making and analyzing tRNA synthetases in vivo and using cell-free protein synthesis. Over two weeks, students will perform site-directed mutagenesis to produce mutant aminoacyl-tRNA synthetases, express their proteins in E. coli cells and purify the proteins, and use cell-free synthesis to attempt incorporating a phosphotyrosine analogue into a target protein using their mutant synthetases and suppressor tRNA. The document outlines the experimental steps, including mutagenesis, transformation, plasmid preparation, sequencing, protein expression and purification, cell-free reaction set up, and analysis by SDS-PAGE. Safety procedures are also described to handle
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This chapter discusses DNA technology techniques including restriction enzymes, recombinant DNA, cloning genes, PCR, electrophoresis, and DNA sequencing. It provides an overview of using bacterial plasmids to clone genes, including restriction digestion to create sticky or blunt ends, ligation to insert DNA fragments into plasmids, transforming bacteria to reproduce the recombinant DNA, and selecting colonies containing the cloned gene. The chapter also describes applications of cloned genes and using restriction enzymes and ligation to create recombinant DNA.
Discover Therapeutic Aptamers For Vegf165 And Egfr
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Streamlined next generation sequencing assay development using a highly multi...
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Next generation sequencing (NGS) assay development for solid tumor sequencing requires characterization of variant calling directly from formalin-fixed paraffin embedded (FFPE) tissue samples. However, cell line based FFPE and human FFPE samples only contain 2 to 20 variants, which require laboratories to invest significant resources in sample sourcing and preparation when developing assays to detect 100+ variantsNannochloropsis gaditana, Master thesis
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This document discusses genome resequencing for SNP discovery and genotyping. It describes what SNPs are, their effects, how many exist in the human genome, and the key steps for SNP discovery including sequencing, validation, screening, and mapping. Methods for SNP discovery discussed include Sanger sequencing, Roche 454 sequencing, Illumina sequencing, and Applied Biosystems SOLiD sequencing.
Circulating Cell Free DNA Targeted Sequencing Workflow | ESHG 2015 Poster PM1...
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Circulating cell free DNA is a potential tumor marker in a non-invasive blood test for the treatment and evaluation of cancer and recurrence monitoring. As circulating tumor DNA is often present at low frequencies within circulating cell free DNA, targeted sequencing on the Ion Torrent™ platform is an optimal tool or mutation detection with very little sample input required. Here, we demonstrate a complete workflow from isolation through molecular characterization of circulating tumor DNA. We have optimized a protocol using magnetic beads to isolate circulating cell free DNA. This protocol is easily automated to process up to 192 samples a day. It is also easily scalable for any input volume and can elute in volumes down to 15 μL resulting in no loss of low frequency alleles. We demonstrate comparable performance between this bead based isolation and column based isolation. We have completed molecular characterization of circulating cell free DNA using the multiplexing capabilities of AmpliSeq™ and the Ion PGM™. With the Ion AmpliSeq™ Cancer Hotspot Panel v2, we performed targeted sequencing of 50 genes of interest, covering 2800 COSMIC mutations. We demonstrate good reproducibility of amplicon representation as well as allelic frequencies. Through saturation studies and subsampling, we have determined the limit of detection of hotspots circulating cell free DNA on the Ion PGM™ to be below 1%. We further demonstrate proof of principle of this workflow on circulating cell free DNA and matched FFPE samples. Our results verify the accuracy and ease of our workflow. This protocol, from isolation through targeted sequencing, will not only result in a simple sample preparation for circulating cell free DNA but also facilitate rapid mutation detection to advance cancer research.Bio Final lab paper
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3) While the pAcGFP1-Mem vector reliably highlights extracellular membranes, its ability to also label some intracellular membranes could be seen as an advantage for experiments examining both membrane types.
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NCSU Poster
This study aimed to clone homologous genes of SND1, a key regulator of secondary cell wall biosynthesis, from Populus trichocarpa. The researcher amplified four SND1 homologs from P. trichocarpa cDNA using PCR. The amplified genes were cloned into E. coli and sequenced. Two colonies were found to contain the correct SND1 sequence insert, while others were false positives. Further work will express and quantify the proteins and determine their effects on other genes and phenotypes.
Chloroplast transformation
Chloroplast transformation allows for the integration of foreign genes into the chloroplast genome. This is beneficial as it provides high levels of transgene expression without epigenetic effects or position effects. Chloroplast transformation requires a chloroplast specific expression vector, a method for DNA delivery such as biolistics, and an efficient selection marker such as spectinomycin resistance. Successful transformation is confirmed by PCR and Southern blot analysis showing integration of the transgene into the chloroplast genome. Applications of chloroplast transformation include production of biopharmaceuticals, vaccines, industrial enzymes, and biomaterials as well as phytoremediation.
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This document describes an improved method for quantitative transcript profiling using cDNA-AFLP (cDNA amplified fragment length polymorphism). The key improvements allow it to be used as an efficient tool for genome-wide expression analysis as an alternative to microarrays. Unique transcript tags are generated from mRNA and screened through selective PCR amplifications. Based on in silico analysis, the enzyme combination BstYI and MseI was chosen to represent at least 60% of transcripts. The method was able to accurately detect differentially expressed genes and subtle expression differences. It was demonstrated to be useful by screening for cell cycle-modulated genes in tobacco.
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The document discusses recombinant protein expression and engineering. It describes:
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2) Factors to consider like the protein's origin (prokaryotic/eukaryotic), required post-translational modifications, and available expression systems.
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This student worksheet contains questions about a pGLO bacteria transformation experiment. It asks the student to define terms related to the experiment such as GFP, transgenic, E. coli, plasmids, ampicillin, and arabinose. It also asks how the student will know if the experiment was successful.
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The Spiller Lab at Rutgers University studies transgenic organisms. Their goal is to create a transgenic bacteria that glows in the dark by taking the GFP gene from jellyfish and inserting it into E. coli bacteria. The GFP gene will be inserted into the bacterial DNA using a plasmid called pGLO. This will allow the bacteria to produce green fluorescent protein and glow, while also conferring resistance to ampicillin, allowing the bacteria to grow on nutrient plates containing the antibiotic.
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1. The document provides instructions for a PGLO lab practice involving transforming E. coli bacteria with plasmid DNA.
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3. Students are asked to cut out descriptions of 12 steps and match them to diagrams showing how to complete the bacterial transformation protocol.
Match ups answers
The document is a worksheet that asks a student to determine which of 10 animals are most closely related by matching them in pairs. It provides the names of 10 animals - African elephant, kangaroo, horseshoe crab, dugong, crocodile, mountain gorilla, ring-tailed lemur, nine-banded armadillo, three-toed sloth, brown recluse spider, emperor penguin, and possum. The student is instructed to match the animals in pairs of the most closely related.
Manatee fact sheet
Manatees are large, slow-moving aquatic mammals that inhabit coastal waters and rivers. They can grow up to 13 feet long and weigh over 1,300 pounds. Despite their massive size, manatees are graceful swimmers that typically glide at 5 miles per hour but can burst up to 15 miles per hour. Manatees give birth underwater and mothers help their calves reach the surface for their first breath. Manatees are herbivores that consume large amounts of water plants. They are classified as endangered due to hunting and accidental collisions with boats.
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Are you interested in the pGLO™ experiment? Learn more about this hands-on laboratory activity with a FREE download of BioRad’s pGLO™ experiment power point.
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The document summarizes steps taken to purify a fluorescent protein tag from a cyanobacteriochrome found in Thermosynechococcus elongatus, including:
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The researcher expressed the wrong fluorescent protein from Thermosynechococcus elongatus, with the pelleted proteins being blue instead of the expected yellow. Through DNA sequencing and comparison to published sequences, they determined the expressed protein was gene 0569 instead of the target gene 0911. Contamination of samples or mislabeling likely occurred. The researcher took steps to verify the correct gene, such as sequencing plasmid DNA from older stock and improving lab techniques, to obtain the expected yellow fluorescent protein in future expressions.
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- 1. Bioengineering a red fluorescent protein tag from cyanobacteriochromes found in Thermosynechococcus elongatus: transfection and visualization using advanced microscopes Mills CBST and Barrett Research Program 2011 Presented by Rosa Meza-Acevedo and Tianling Ou
- 2. Our Research Goal Bioengineer a new red fluorescent tag Red illumination of the cytoskeleton Images modified from: cbst.ucdavis.edu
- 3. Genes from CyanobacteriochromesT. elongatus
- 4. Review of our procedure
- 5. Our Focus
- 9. ideal to study HIV viral entry
- 11. Maintenance:
- 12. CO2 incubator
- 13. Optimal cell density: 30*104 cells/ml
- 14. Selection:
- 15. G418 antibiotics
- 19. Centrifugation and Aspiration to collect the cells in a pellet
- 20. Resuspend the pellet with transfection reagent, and mix with plasmids. Transfer to a special nucleofectioncuvette.
- 25. DNA purity
- 27. Endotoxin-free
- 28. Low passage numbers of Jurkat Cells
- 30. A video from CBST, taken on a deconvolution microscope
- 31. GFP transfected cells pLifeAct-TagGFP2 electroporation pAc-GFP-Tubulinelectroporation
- 34. Barrett Family
- 35. Deanna Thompson and Ana Popovic from CBST
- 36. UC Davis Medical Center
- 37. Dr. Lagarias and Nathan Rockwell
- 38. Mills CBST lab members
- 39. National Science Foundation (NSF)Thank you
Editor's Notes
- Add a red fluorescent tag to the cytoskeleton of a lymphocyte to …Our research goal is to bioengineer a new red fluorescent tag. We want to do it because the red fluorescent tag can be a very useful tool to help us visualize living cells under advanced microscope. Here you can see the picture. This is a cell with red labeled cytoskeleton. Cytoskeleton is the internal network of the cell. On the top, it’s HIV labeled by green fluorescence, and a healthy Jurkat cell labeled by red fluorescence. Jurkat cell is a lymphocyte cell in the immune system.
- PCR genomic DNA with primers to capture the GAF domain DNA sequence Insert our PCR product into a transformation vector built upon pBAD by doing restriction enzyme digest and ligation Site directed mutagenesis to change amino acid cysteine in the class II GAF motif to aspartate to make it fluorescence Transform E. coli bacteria with two plasmids pBAD + insert and pPL from Lagarias protein expression in the E. coli system using the plasmids we constructed protein purification to confirm the fluorescence (Wavelength, brightness)
- pDsRed-Monomer-Actin Vector: This plasmid encodes DsRed-Monomer, a monomeric mutant of the Discosoma sp. red fluorescent protein DsRed. The pDsRed-Monomer-Actin fusion protein incorporates into growing actin filaments, allowing visualization of the actin cytoskeleton in living or fixed cells.This plasmid contains all the correct origins of replication and promoter regions to be active in both e.coli and mammalian cells. G418- antibiotic mixture – Neo, allowing the selection of Jurkat cells that actually incorporated the plasmids.
- INSERT a picture of Jurkat
- The basic mechanism of transfection (using plasmid DsRedMonomer-Actin as an example):The DNA combines with transfection reagent. The complex will then enter the cell through the transient pores. Chemical method and physical method for opening the pores
- The difference between chemical method and electroporationUse of high-voltage electric pulse to perturb the cell membrane and form transient pores, introducing DNAHighly efficient for the introduction of plasmids, especially mammalian cells Process:Cell Counts: an accurate cell concentration is very essential for electroporaion. One million cells per millimeter is required.Cells are extremely fragile after nucleofection, because there are pores in the membrane.Sterile technique is strictly demanded
- Our data is not sufficient enough to draw a valid conclusion. We are looking forward to a cell culture room at Mills, so that we can continue and optimize the transfection of Jurkat cells. The estimated concentrations of these plasmids may not be accurate pAcGFP1-tubulin turned out to be more concentrated when measured by nanodrop
- You may wonder what gel quantification and nanodrop are.Gel quantification is to run a DNA gel with our plasmids and markers. You can see here the bands on the gel. We compared the brightness and position of the plasmids to the markers. Since we know the size and concentration of each band of the markers, so that we can estimate the concentration
- The Deconvolution fluorescence microscope at CBST is used for rapid live and fixed cell fluorescence microscopy. It can capture time elapse images showing the fluorescent cytoskeleton in 2 to 3 dimensions. Dark and temperature controlled
- This is the video from CBST of Jurkat cells that transfected by pDsRed-Monomer-Actin. The two Jurkat cells are ~15um in diameter. This video was taken on a deconvolution microscope.The grey cells that appear in the end are also Jurkat cells, but are not transfected successfully, and thus they are not fluorescent.