...components into mammalian cell lines is quite broad and includes several different methods, so we've summarized...

...experimental design. Search Addgene's collection of empty LIC cloning vectors Protocol Step 1: Design Your Primers... Primers Primer design for LIC is often as simple as using the backbone manufacturer's suggested leader...primer is shown here. Note: Use web-based primer design software to ensure a melting temperature between...nucleotide dGTP in the reaction (exclude all other nucleotides from standard polymerase protocol), causing the...manufacturer. It is very important to remove all free nucleotides from your PCR product before proceeding, as they...

... to a new plate containing the mastermix which includes primers and a ddPCR supermix. Note that the supermix...

...minigenome system Minigenome systems, such as the one described here, are extremely useful research tools. In ...

...activation. Including a prey-only control also provides a baseline level of reporter activation. Vary ...

...that extra two miles which is way more fun. And despite those who may wish to tell you so – towel corners...

... plasmid delivery to a cell. In contrast, TREE provides a read out for plasmid delivery and subsequent...

...collaborators than you realize. Being at a university provides the perfect platform for practicing initiative...

... this re-inversion issue. It became possible to design FLEx vectors (Flip-excision vectors) that only ...

...will identify a ‘scientific mentor’: someone who provides career advice and offers direction on your scientific...

...an abasic site. Like in most other base editor designs, the Cas9 nicks the non-edited strand to encourage...

...plasmid of interest. For this example, we will describe how to copy a cDNA from one vector into a new ...(YGOI) for ligation into a recipient plasmid. Designing Primers for PCR Based Cloning The basic PCR primers...that: Do not cut within your insert. Are in the desired location in your recipient plasmid (usually in ...examine the DNA sequence that we want to amplify and design primers that will bind to and replicate it. The...ends of the ORF and how these are used for primer design: Because we are cloning an ORF, we want to clone...TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design is similar, but we need to use the reverse complement...

...This protocol describes the basic steps for lysing cells, determining total protein concentration in ...specific to the protein of interest. This protocol describes the basic steps for lysing cells, determining ...continue running the gel until you have obtained the desired separation. Gently open the gel with the spatula...Device. Close the lid of the device. Select the desired method and make sure the parameters are correct... incubation Dilute the primary antibody to the desired concentration in blocking buffer. Pro-Tip The optimal...peroxidase-conjugated secondary antibody to the desired concentration in blocking buffer. Pro-Tip The optimal...

...primer should be a minimum of 50 nucleotides and a maximum of 300 nucleotides from your target. Are there ...Analysis of a Plasmid You may also like... How to Design a Primer Diagnostic Restriction Digest Introduction...If you require additional sequencing and need to design a custom primer, Addgene recommends using Addgene's...'s sequencing results as a reference for primer design. A good sequencing reaction will produce between...

... of bacteria or fungi. There are disinfectants designed to be used in water baths with instructions on...bath on and set the appropriate temperature as described in your protocol. How to set the temperature will...Set up your water bath so that it will reach the desired temperature by the time you need it for your experiment...water bath to prevent evaporation and maintain the desired temperature. This also helps the water bath heat...water bath even if you are using disinfectants as described above in step 3. You will also need to maintain...

...This protocol describes the basic steps for fixing and staining cells in culture with a primary antibody...antibodies to detect antigens in cells. Here we describe the basic steps for fixing and labeling cells ... per well. Allow the HeLa cells to grow to the desired density before labeling. Section 2: Fixing and ... container. Dilute the primary antibody to the desired concentration in antibody dilution buffer. Pro-...fluorescently-labeled secondary antibody to the desired concentration in antibody dilution buffer. Pro-...

...even better. The Thomas Kilmann Conflict Model describes 5 basic approaches to conflict resolution based...

...Biol 24, 758-766 e753 (2017). Baloban, M. et al. Designing brighter near-infrared fluorescent proteins: insights...

...with a site mutation (R11G). This site mutation destabilizes the ligase, allowing for active biotin molecules...

...if I had planned my day extensively, my lack of desire to go sit in the tissue culture hood may have prevented...