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... 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. (Link opens in a new window...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...

...Quantify virus by counting antibiotic resistant colonies Generating Stable Cell Lines with Lentivirus Genomically...Introductory techniques designed to help you get started in the lab. Name Description (Link opens in a new...monoclonal cell lines from a polyclonal pool of stable cells AAV Production in HEK293 Cells Produce adeno-associated...Protein G columns Watch the Video! Coomassie Purity Stain Determine purity and concentration of recombinant...

... carry one or more antibiotic resistance genes, which confer resistance to a specific antibiotic to the... carrying them. The presence of an antibiotic resistance gene on a plasmid allows researchers to easily...intend to do a miniprep, you will usually want to start 2 mL in a Falcon tube. For larger preps, you might...antibiotic in your LB media matches the antibiotic resistance on your plasmid. If the bacteria on your LB agar...

...for a variety of downstream applications such as stable-cell line generation. Workflow Timeline Day 0: ... 10-013-CV 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...and for each cell line. Considerations Before You Start The health of the packaging cell line is critical...endonuclease negative E. coli strain such as NEB stable. Make a mixture of a total of 500 μL PEI-OptiPro...

...Standard protocol for performing agarose gel electrophoresis, including tips to improve resolution and...Ligation Introduction Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g....New England Biolabs B7022S Procedure Pouring a Standard 1% Agarose Gel: Measure 1 g of agarose. Pro-Tip... solution and rinse with water to even out the staining after the gel has been run, just as you would ...-30 mins, replace EtBr solution with water and destain for 5 mins. Using any device that has UV light,...

...purification step, it is important to digest plenty of starting material. We recommend 1.5-2μg of donor plasmid...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...instructions for your competent cells. For most standard cloning, you can transform 1-2μl of your ligation...

...containing the carbenicillin (amp) resistance gene (or other antibiotic resistance) Procedure Day 1 Prepare carbenicillin... cells containing plasmids differing in their resistance genes, as one does not need to prepare separate...place of ampicillin because carbenicillin is more stable, so it is potentially more effective at selecting...

... out more protocols and videos to help you get started in the lab! Introduction For many experiments, ...precisely to create the solution you expect. Understanding how to obtain the correct amounts of materials...weighing and prevent cross-contamination with other substances. They also help you transfer the material to ...your lab’s material safety data sheets (MSDS) to understand how to properly dispose of reagents that you ...

... use it to allow the temperature to stabilize. Use water resistant markers, such as permanent markers,... out more protocols and videos to help you get started in the lab! Introduction A water bath is a piece...water bath, be sure to use a marker that is also resistant to chemicals such as ethanol. Writing on the tops...

...Dam or Dcm methylase positive strains will be resistant to cleavage at certain restriction sites. See ...identical, to their recognition sites. This is due to "Star Activity" and can happen for a variety of reasons...(Link opens in a new window) NEB's website about star activity . If you are digesting a large number of... of plasmids with the same enzyme(s) (for instance, in a diagnostic digest), you can create a "Master ...

... 10-013-CV 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... a new working stock. Considerations Before You Start The health of the cell line is critical for obtaining...endonuclease negative E. coli strain such as NEB stable. Dilute 1:3 (µg DNA:µg PEI) in 500 µL total of ...

...the two ends of the vector together). Protocol: Standard Insert + Vector DNA Ligation Before setting up...each and their concentration. However, for most standard cloning (where the insert is smaller than the ...fine. We recommend around 100ng of total DNA in a standard ligation reaction. Use a (Link opens in a new ...of competent cells and verifies the antibiotic resistance of the plasmid Cut vector - Background due to...

...protocol describes the basic steps for fixing and staining cells in culture with a primary antibody against...15 mL conical tubes 50 mL conical tubes Before Starting Refer to the manufacturer's instructions for additional...500 µL PBS on a rocking platform. (Optional) Counterstain nuclei with 500 µL of 300 nM DAPI working solution... Review the manufacturer's instructions before starting your experiment and consider titrating your antibody...