...the cell density and viability: Using a 5 mL pipette, transfer 0.5 mL of HEK293 cell suspension into a ...microcentrifuge tube. Vortex the cell suspension. Transfer 10 µL of cell suspension into the microfuge tube containing...Protocol Introduction Transfections allow for transient expression of a gene of interest in cell culture...culture. This protocol describes how to transfect suspension HEK293 cells with recombinant antibody plasmids...support reproducibility in science. General Considerations For antibody-expressing plasmids containing...antigen (EBNA). Safety Warnings HEK293 cells are considered biosafety level 2. Please ensure that you are...transfecting, seed a 108 mL culture of HEK293 cells at a density of 0.9 x 10 6 cells/mL in a 500 mL vented flask.Incubate...

... short-term protein expression observed using transient transfection approaches, generating cell lines...experiments in a stable cell line, as opposed to transiently-transfected cells, increases reproducibility,...eliminates the variation associated with repeated transient transfection. Some lentiviral vectors deliver ... in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health of the target cell...pipetting or inverting the tube. Aliquot 1 mL of cell suspension (i.e., 50,000 cells) into each well of the 6-...the virus will have higher levels of expression. Consider expanding populations transduced with a variety...expressors may take over the culture. To overcome this, consider generating monoclonal lines from the early polyclonal...

...steps 2-4 25-30 times. Final Extension for 5 minutes at 72°C: A final extension to fill-in any protruding...below the primer melting temperature (Tm). Set extension step at 1-2 minutes per kilobase of product depending... instructions for specific instructions about extension time and temperatures. Initial Denaturation for...°C. Repeat steps 8-10 for 25-30 cycles. Final Extension for 5 minutes at 72°C. Run 2 μL on a gel to check...The rate of DNA synthesis is ~1-2 kb/min. The extension time can be adjusted according to the length of... temperature around 70°C and is the molecule responsible for driving the DNA synthesis. Sterile dH 2 O...

...culture of bacteria transformed with your plasmid Resuspension buffer Denaturing solution Renaturing solution... This step gets all of the bacteria back into suspension, but within a smaller volume of buffer that is...Prepare the following solutions: Solution I - Resuspension Buffer 25 mM Tris-HCl (pH 8) 50 mM glucose 10...phase Middle phase - A white layer may appear, consisting of precipitated protein particles Bottom phase... mM Tris-HCl pH 8, 0.1 mM EDTA). Pro-Tip DNA resuspension can take time, it is a good idea to let it sit...eliminate RNA contamination. This is included in the resuspension buffer of most kits. Reference Page Top Index...

...monoclonal line could be generated at any time. Considerations Before You Start Some cell lines tend not to...optimized for adherent cells but can be modified for suspension cells. The number of monoclonal lines generated... this, you are seeding the plate at an average density of 0.5 cells/well. Seeding an average of 0.5 cells...Figure 1: Generation of monoclonal cell lines from expansion of individual A549 cells stably expressing Cas9... in individual wells of a 96-well plate after expansion of single cells for 13 days. Note that growth ...stable cell pool (pool) and from individual cell expansions (monoclonal, 1-5). Wild-type A549 cells do not...

...liquid culture is capable of supporting a higher density of bacteria and is used to grow up sufficient numbers...need to grow for a longer time to get the correct density of bacteria since they will grow more slowly at...specific protocol and conduct an OD600 to measure the density of your culture if needed. A good negative control...culture. More aeration may help to increase the density of the culture. Normally cultures shake at 150 ... this to 350 - 400 rpm to obtain a higher cell density. Reference Page Top Index...

...applications. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please ensure that you are... in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health of the target cell... pipetting or inverting. Aliquot 2 mL of cell suspension into each well of the 6-well dish. Incubate the...anticipate that your viral stock will be higher titer, consider additional dilutions. Dilution Volume of Lentivirus...cells in each well. When calculating titer, only consider wells with less than 40% fluorescent cells. Titering...

...tissues, extraction of RNA, precipitation, and resuspension. This protocol provides two options: using Solution...phase without disturbing the interphase layer, consider using a lower volume pipettor like a p200 to collect... freeze-thaw cycles of your entire RNA sample, consider making smaller aliquots of it and storing those...

...solution or a 70% ethanol solution to wipe down the inside of the water bath. Pro-Tip To properly maintain...maximum fill capacity is. Look for a “fill” line inside the water bath or read the user manual. If you’...may float in the water bath when they are placed inside. Water bath floats can be used to secure tubes ...before use, make sure that there is enough water inside....

...Addgene's online resources. Career Videos Get career insight and advice from real Addgenies! Protocols (Link... once using a multichannel pipette and avoid inconsistent pipetting with our simple tips Bacterial Transformations...Australia, and advice for current graduate students considering their future careers. Eric J. Perkins, PhD In...Addgene's Business Development Manager, discusses his transition from the Scientist Team to Business Team, his...

... check to make sure that it isn't methylation sensitive . Plasmids grown in Dam or Dcm methylase...opens in a new window) NEB's table of methylation sensitive restriction sites . Sometimes enzymes cut sequences...diagnostic digest), you can create a "Master Mix" consisting of all of the reaction components except for ...each tube. This will save you time and ensure consistency across the reactions....

...1 mL). When working in a laboratory, properly dispensing liquid ensures the accuracy of your experiment...experiment and any changes to the amount you are dispensing can negatively impact your experimental results. ...the liquid and avoid touching the pipette to the inside of the tube. Only the tip should be in the liquid...different tubes or into wells of a microplate, you may consider using a multichannel pipette. Please watch Addgene...

... 10% bleach. Safety Warnings AAV is generally considered biosafety level 1 but may require BSL-2 handling...45° angle. Count to 20 while slowly and gently dispensing the droplets down the side of the tube. Place... 95 10 2 1 Denaturation 95 0.5 2 50 Annealing/Extension 60 1 2 50 Signal Stabilization 98 10 2 1 Hold ... should be close to zero (B08). At Addgene, we consider runs with an NTC > 10 to be invalid. To reduce...

... through a 0.22 μm membrane. Store at 4 °C. Considerations Before You Start The health of the HEK293T ...vigorously multiple times to obtain a single cell suspension (no clumps of cells). Pool cells from 2 x T-175...forth or shake vigorously to obtain a single cell suspension (no clumps). Count cells using a hemocytometer...above. Add 50 units of benzonase per mL of viral suspension (you should have ~18 mL from steps 12 & 13). ...

...bleach. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please ensure that you are... media and 11.1 µg/mL Polybrene. Mix the cell suspension well before seeding. Mix each well with a 1 mL...45° angle. Count to 20 while slowly and gently dispensing the droplets down the side of the tube. Place... 95 10 2 1 Denaturation 94 0.5 2 40 Annealing/Extension 60 1 2 40 Enzyme Deactivation 98 10 2 1 Hold 4...

... in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health of the target cell...pipetting or inverting. Aliquot 1.35 mL of the cell suspension into each well of the 6-well dish directly on... 48–72 h. Pro-Tip Different cells may be more sensitive to lentivirus or to polybrene, in which case this...

... In research, iodixanol is used as an isomolar density gradient medium suitable for virus purification...to mammalian cells. Therefore, and unlike other density gradient media, it is reportedly not necessary ...steps are used to remove contaminants with lower densities, including empty capsids. The 60% step serves ...

..., be aware that some gloves may contain latex. Consider nitrile gloves as an alternative. For extra precautions...anyone else associated with Addgene, can be held responsible for your use of the information contained in ...

...satellite colonies will grow). It is, however, more expensive. With a 6 cm diameter petri plate containing solidified...Plate 50 µL of transformed E. coli /rescue media suspension onto the agar and gently spread over the surface...

...from a liquid or separate liquids of different densities is essential to many different experimental procedures...samples. If your samples are significantly higher density than water, then you may need to fill the blank...