... Protocols Generating Stable Cell Lines with Lentivirus Generating Stable Cell Lines with Lentivirus... antibiotic resistance (e.g., puromycin, blasticidin), which enables selection of a stable cell culture...lentiviral vectors deliver antibiotic resistance. This protocol was established using 293T cells but can be adapted... of antibiotic, which may not be stable at 37 °C. To achieve a stable cell pool, the antibiotic selection...expression. As polyclonal populations of resistant cells start proliferating and the individual wells become...Protocol to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector...Introduction This protocol can be used to generate stable cell lines expressing a gene of interest from an...

...help stabilize the standards add carrier DNA to a final concentration of 4 ug/mL to each standard dilution... Once a validated standard curve is obtained, make a small aliquot of each standard (enough for 1 or 2...their initial value. If the Ct value of the standard starts to drift, it’s time to make a new one. When...your plasmid standard with DNase ** Make 6 serial dilutions, in duplicate, of your standard curve plasmid...5'-CGGCCTCAGTGAGCGA ITR-containing plasmid for standard curve RNase-free DNase 10X DNase buffer Nuclease-free...Pipette tips General Considerations Always run standards and samples in duplicate at least If possible,... the samples have been added to the qPCR plate. Start by adding water, then SYBR master mix, then the ...

... carry one or more antibiotic resistance genes, which confer resistance to a specific antibiotic to the...to be resistant to the antibiotic. On the second plate, streak out a strain that’s not resistant to the... be resistant to the antibiotic, (+) indicates that the tested strain is supposed to be resistant to the...Negative Result 3: Only the Non-resistant Strain Grows If only the non-resistant strain grows, it’s likely ... carrying them. The presence of an antibiotic resistance gene on a plasmid allows researchers to easily...for the selection of only those bacteria with resistance to that antibiotic - usually conferred by a plasmid... plasmid carrying the antibiotic resistance gene. The following protocol will allow you to make your own...

...transfer stack. Set the Top Stack to one side and discard the white separator. Keep the Bottom Stack in the...SDS-PAGE running buffer Prestained protein ladder Ethanol iBlot 2 PVDF Mini Stack, Thermo Fisher IB24002... of the BSA standard that range from 0–2000 µg/mL . In duplicate, dilute 10 µL of standard, blank, and...blank from all of the samples. Plot a standard curve of BSA standard concentration versus absorbance. Extrapolate...antibody Secondary antibody Deionized water Before Starting Refer to the manufacturer's instructions for additional...total protein concentration of the sample from the standard curve. Determine the volume of sample required...the samples into the gel. Load 5–10 µL of the prestained protein ladder. Place the lid on the tank and...

...markers. Note this assay requires staining resistant colonies with crystal violet solution and, therefore...Change media as needed Days 14–18: Stain cells and count resistant colonies Equipment Class II, Type A2...Filter 0.1% crystal violet solution through a 0.22 μm filter to remove any precipitates. Stain each well...well with 1 mL of 0.1% crystal violet for 10 min at RT. Gently remove the stain. Wash cells 3x with 1 mL...exchanges every 3–4 days. Colonies were then stained with 0.1% crystal violet and counted. Figure 2: A549 cells...lentiviral preparations that confer antibiotic resistance. Protocols...lentivirus preparations that confer antibiotic resistance. Determining the titer of your lentiviral preparation...

...protocol begins with a stable cell pool. For details, see our protocol for generating stable cell lines with ...Store at 4 °C. Polyclonal stable cell pool: see our protocol for generating stable cell lines with lentivirus... monoclonal cell line from a polyclonal pool of stable cells. Protocols... monoclonal cell line from a polyclonal pool of stable cells. Transducing cells with lentivirus results...cell populations that are more likely to retain stable transgene expression. Other methods of generating...Hemocytometer or other cell counter Reagents Polyclonal stable cell pool DMEM high glucose, Corning 10-013-CV ...CV L-alanyl-L-glutamine (or alternative stable glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated...

...power objective is directly over the stage. It is best practice to start with the lowest power objective to...microscope stage. If using a slide, you can secure it into place using the metal clips on the stage. Turn ...Turn on the power source and use the stage arm to move the stage so that the light shines onto your sample...Once your sample is in focus, use the stage arm to move the stage (and ultimately your slide) until your... out more protocols and videos to help you get started in the lab! Introduction Microscopes are emblematic....com. Base Light Source Condenser and Diaphragm Stage Objective Lenses Focus Knobs (Fine and Coarse) Nosepiece...of light hitting the sample or b) using dyes or stains that add color to some components of the sample...

...introduced, the puromycin resistance marker encoded in pLKO.1 allows for convenient stable selection. Figure ...with effective gene silencing: Starting at 25nt downstream of the start codon (ATG), search for 21nt sequences...homologous recombination of unstable regions, such as Invitrogen Stbl3™ or NEB Stable cells. This will ensure...Stock solution is stable at 4°C for up to one year. The powder form of polybrene is stable at 4°C for several...drives expression of puromycin. Puro R Puromycin resistance gene for selection of pLKO.1 plasmid in mammalian...bacterial origin of replication. Amp R Ampicillin resistance gene for selection of pLKO.1 plasmid in bacterial...should be 36-52%. Sense 3’ end should have low stability – at least one A or T between position 15-19. ...

...Plasmid DNA DNA Ligation Background Information A standard Polymerase Chain Reaction (PCR) is an in vitro...became automated with the discovery of a heat resistant DNA polymerase from the thermophilic bacterium... Thermus aquaticus (Taq) . Taq polymerase can withstand many heating and cooling cycles, which would denature... template strand to the point where the strands start denaturing and the hydrogen bonds are broken between...seconds at 55°C: The forward and reverse primers are stable within this temperature range to anneal to each...DNA template strands. The DNA polymerase is also stable enough to now bind to the primer DNA sequence. ...Taq Buffer with MgCl 2 : Provides an optimal and stable chemical environment for the DNA polymerase to ...

...Before Starting Warm reagents to room temperature. Procedure Section 1: Prepare the Antigen Standard and ...suggested starting point. If your unknown sample’s absorbance falls above the range of the standard curve ...antibody. This protocol will describe how to create a standard curve using known concentrations of the antigen...first three wells of each row, using a different standard solution for each row of a 96-well ELISA microplate...needs to be empirically determined. We suggest starting with three concentrations between 1–10 µg/mL. ...spectrophotometer. Use the spectrophotometer to generate a standard curve . If you don’t see any color change or dose...antibody concentrations. The ideal range of the standard curve will vary between targets and needs to be...

...Culture Introduction If you have a glycerol stock or stab culture of bacteria and need to purify plasmid DNA...agar plate (with appropriate antibiotic) Bacterial stab Procedure Obtain an LB agar plate with appropriate... It is also a good idea to add the antibiotic resistance and your initials. Labeling within a laboratory...organization, and it is recommended that you keep a standard labeling system for all your objects/solutions...bunsen burner. Obtain the approrpriate bacterial stab or glycerol stock . Using a sterile loop, pipette...bacteria growing within the punctured area of the stab culture or the top of the glycerol stock. Pro-Tip...popular technique is to draw in discontinuous lines. Start by streaking a vertical line of bacteria along one...

...regions (usually 4 bp) does not provide enough stability to hold the plasmid together through the transformation...replication process. LIC employs long overhangs to form a stable association between fragments, allowing for transformation... enzymes (e.g. BsaI), which cut at a specified distance from their recognition sequence. This can create...fused to your gene of interest, in frame with the start codon or tag sequences (where appropriate). The ... which point it will add back the G and become stalled. Therefore, the primer must begin with the following...the reaction (exclude all other nucleotides from standard polymerase protocol), causing the enzyme to perform...will add back the guanosine residue and become stalled. A typical T4 Pol reaction is shown. Mix the components...

... where these are located before you start. Use the eyewash station if unwanted or biohazardous materials... out more protocols and videos to help you get started in the lab! Introduction There are four biosafety... with pathogens including organisms such as Staphylococcus aureus or Vibrio cholerae . BSL-2 includes ..., or apply makeup in the laboratory. Before you start your experiment, make sure your workspace is clean...contact with hazardous materials. A sink, eyewash station, safety shower, fire blanket, and extinguisher ...received hazardous waste safety training before starting the work. Before working with chemicals, first...receives a hepatitis B vaccination or titer prior to starting work in the laboratory. For some biohazardous ...

...flask, Corning 430825, 175 cm 2 Cellstack 5, Corning 3319, 3180 cm 2 Cellstack 2, Corning 3269, 1272 cm 2 ...used to produce AAV from one Five Chambers Cell-Stack (CS5) (Link opens in a new window) (3,180 cm 2 -...the same surface area as 21 x T-175 flasks). Cell stacks provide an efficient means to scale-up without ...(optional) L-alanyl-L-glutamine (or alternative stable glutamine such as glutaGRO, Corning 25-015-CI) ...: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or stable alternative, such as glutaGRO) To a 500 mL bottle...membrane. Store at 4 °C. Considerations Before You Start The health of the HEK293T cells is critical for ... Sonicate until no live cells can be seen when stained with Trypan Blue. Pellet cell debris by centrifugation...

...we are cloning an ORF, we want to clone from the start codon (ATG) to the stop codon (TGA, in this example...give specificity and to also be compatible with a standard PCR reaction. Therefore, our Forward Primer will...reverse complement to get PCR amplification. We can start similarly, taking the final 18bases of the ORF, ...purification step, it is important to digest plenty of starting material. We recommend using your entire PCR reaction...lanes between samples. In addition to a DNA ladder standard, it is also a good idea to run an uncut sample...plasmid. We recommend around 100ng of total DNA in a standard ligation reaction. You ideally want a recipient...important to set up negative controls in parallel. For instance, a ligation of the recipient plasmid DNA without...

... for Ampicillin resistance but is much more important for other antibiotic resistances. Plate some or ...bacterial origin of replication and an antibiotic resistance gene for use as a selectable marker in bacteria...colonies. Equipment Shaking incubator at 37 °C Stationary incubator at 37 °C Water bath at 42 °C Ice bucket... the bacteria time to generate the antibiotic resistance proteins encoded in the plasmid backbone so that...Shorten or skip the outgrowth (for Ampicillin resistance it is ok to completely skip the outgrowth, for... plate containing the correct antibiotic. The resistance gene on your plasmid must match the antibiotic...

...idea to assay each fraction by silver stain or SYPRO Ruby stain to determine purity. Only the cleanest...preparation. The 15% iodixanol step has 1M NaCl to destabilize ionic interactions between macromolecules. The...QuickSeal tubes out of the rotor and place them in a stable rack. **Make sure not to disturb the gradient!*...top of the QuickSeal tube with a 16 ga needle and start collecting 0.5 mL to 1 mL fractions per tube. Avoid...at the bottom using an 18 ga needle. Immediately start collecting 0.5 to 1 mL fractions in microcentrifuge...consecutive gradient fractions followed by silver stain. Note the increased number of contaminants in each...

... of a substance in that liquid. In order to accurately measure the concentration of a substance based ...Background Information During several different stages of molecular cloning, it is important to get a ...to determine the concentration of a particular substance in that liquid. Molecules absorb different wavelengths...need to know the wavelength of light that your substance maximally absorbs. In the case of nucleic acid...samples, place 1-2µL of mini-prepped DNA onto the pedestal. Close the lid and click measure, be sure to record...

...questions. Protocols Video protocol guides for standard laboratory procedures. How-To Videos Screencapture...Bacterial Transformations We walk you through a standard plasmid transformation protocol and offer some...Inoculating a Liquid Bacterial Culture Protocol Getting Started with Tissue Culture Tips and tricks for working...Gel Electrophoresis Gel electrophoresis is the standard laboratory procedure for separating DNA by size...Description Jessica Welch, PhD In this first installment of the Addgene Careers series, we sit down with...future careers. Eric J. Perkins, PhD In this installment, we sit down with Senior Scientific Project Leader...

...time, you will need to establish glycerol stocks. The addition of glycerol stabilizes the frozen bacteria...alive. A glycerol stock of bacteria can be stored stably at -80°C for many years. Protocol Video Watch the... glycerol stock tube at -80°C. The stock is now stable for years, as long as it is kept at -80°C. Subsequent...information page. The next day you will be able to start an overnight culture for plasmid DNA prep the following...