...transforming large plasmids (>10 kb) or BACs, what can I do? Chemically competent cells are fast and easy to ...cell/DNA mixture to induce membrane permeability. To do this you will need to have access to an electroporator...

...box and push the end of the pipette onto the tip. Do not touch the tips with your fingers to avoid contaminating...plunger, gently lower the pipette tip into the liquid. Do not submerge the pipette itself into the liquid and...

...regularly Do not over or under-grow your cells. Thaw a new vial of cells after 20–30 passages. Do not add...Procedure Before beginning a colony formation assay, the dose of antibiotic required to kill your target cell ...determined. Treat the target cells with a range of doses of antibiotic. Determine the minimum concentration...window) required to kill all of the cells. Use this dose for the colony formation assay. Prepare a batch ...

...thicker as the proteins and DNA are denatured. Pro-Tip Do not vortex at this stage or the genomic DNA will ...4. Note: Phenol-chloroform is a hazardous waste - DO NOT pour down sink. Top Protocol: Ethanol Precipitation...

...Media 10 mL 10% Pluronic-F68 40 mL 200 mM Glutagro Do not add selective reagents Store at 4 °C until use...incubator on a shaking platform set to 120 rpm. Pro-Tip Do not use cells that are over 30 passages. Section ...

...complementary to template strand). However, primers do not need to correspond to the template strand completely...

...scrape some of the frozen bacteria off of the top. Do not let the glycerol stock unthaw! Streak the bacteria...

...surface. Tare the weighing boat or paper. You need to do this because you don’t want to include the weight...

...entire dilution plate with Microseal adhesive seal - do not press the film, just gently cover so nothing ...collecting drops along the side of the tube. Cap gently - do not push the cap in all the way, just ensure the ...

...the surface). We use the micropipette tip itself to do the spreading; the tip is gently bent to create an...

...of the tube. This way, as items are floating, you do not need to necessarily maneuver the bottles or tubes...

...the vector with EcoRI and SalI (see diagram ). To do this, we add 5' - AATTC and G - 3' to the top oligo...

...Protocol Storing and Handling Addgene Plasmids What to do after you receive your plasmids from Addgene Instructions...

... centrifuge and be dangerous for the user. If you do not have a balanced number of samples, you may need...

...images directly to a computer, but many microscopes do not and you may have to dust off your drawing skills...

... and a negative control sample such as cells that do not express the protein of interest....titrating your antibody to determine the optimal dose. To ensure that your antibody is both functioning...

...8x10 5 cells in a T75 flask in a volume of 15 mL. Do not add antibiotics to the media. Use cells that ...

...10 5 cells in a T75 flask in 15 mL DMEM Complete. Do not add pen-strep to the media. Use cells that are...

...sample from the reservoir to a LoBind tube. Pro-Tip Do not scrape the filter with the pipette tip. Determine...

...purified antigen to demonstrate the expected antibody dose response....probe with an antibody against the target to show a dose response. Sharing speeds science. We believe that...standard curve . If you don’t see any color change or dose response, your antibody concentration may have been... the reaction is saturated and you don't see any dose response, your antibody concentration may have been...