...theory or practice; business-like or artistic; tech, anti-tech or pragmatic.” Advice for pursuing careers...

...paint your cells in equally vivid colors, you'll want to check out the 34 new preps available in the Addgene...

...museums can be excellent methods to detach from the constant stream of thoughts related to lab research and...

...for plasmid quality control are: To identify important plasmid features To align NGS sequencing results...

...members to make sure the courses encompass what they want to teach.” Besides working with students and faculty...

...GCN4. GCN4 recruits GFP fused to the cognate scFV antibody, which is expressed from a separate plasmid. This...

...daily challenges a little easier to overcome. We want to encourage researchers all around the world by...

...doing, managing or leading. All three roles are important to accomplishing any goal or finishing any project...

...problems. While giving corrective feedback isn’t pleasant, it’s far worse for employees if you don’t care...

...beware of not micromanaging the scientist if you want their best work) Are continuous learners and have...

...discarded.  Seeding Labs does an amazing job shuttling unwanted lab supplies to places that really need them.  ...

... et. al, 2013 Science paper. For example, if you want to use double nickase, you could express two spacers...

...neuronal circuit tracing.     Marcus Leiwe is an assistant professor at Kyushu University. He has a particular...

...So you have this awesome experiment you want to do, but it requires some AAV. You’ve never worked with...

...tagging two cell populations in a competitive transplant assay in the mouse intestine, demonstrating its...

...blog, or our Viral Vectors 101 eBook, out if you want to learn more. References and resources References...

...educational resources and experience to their advantage should they decide to produce virus on their own.Harvard...

... of genome editing and human reproduction, the relevant document for the question of “how do we move forward...

...sequence replacement holds some kind of fitness advantage but error-prone repair of the gene would be disastrous...

...burdensome task of explaining the steps to a lab assistant. However, I recognized the need to shift my mindset...

...is not currently known to cause any diseases. Recombinant AAV largely does not integrate, and the addition...

...This table is by no means exhaustive, so you may want to consult the REBASE database for more detailed...

...patient mutations using SDM can help determine if a variant is causal for a given disease. CRISPR has made ...

...Averaging ten panels each. (For this story, it is important to know that the student presenting right before...

...tough assignments, the skills that Susan  finds important for her work include: “Listening. Evaluating evidence...

...biology known as bioelectricity is revealing the important role of bioelectrical signaling mediated by ion-channels...

...talk. Some video conferencing software allows participants to “raise” their hand to ask a question or type...

...muscle groups (usually the thigh), though it is important to avoid injection into the sciatic nerve. Mice...

...function in the human body?” … and probably most importantly… "how do we know this?” Over the past fifteen...

...simple to use, users should keep aware of a few important details. For transposons to be specifically recognized...

...cloning strain has failed so far. What is most important is the transformation efficiency of the strain...

...newly generated MCS. MXS-chaining also has the advantage of being directional, meaning that (in the previous...

...that company culture and enjoying your job were important parts of team buidling - they relieved some of...

...experimenting with open source assembly algorithms meant that we already had a clear idea of the necessary...

... scientist position at Addgene, the skeptical applicant asked if she would partake in “critical thinking...

...D.A., et al., Generation, purification and transplantation of photoreceptors derived from human induced...

...right for appropriate antibiotic concentrations. Antibiotic Concentrations Antibiotic Recommended Stock Concentration...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...that: The antibiotic broke down. You forgot to add the antibiotic. You added the antibiotic at too low... any antibiotic. Negative Result 3: Only the Non-resistant Strain Grows If only the non-resistant strain...one or more antibiotic resistance genes, which confer resistance to a specific antibiotic to the bacteria...resistance to that antibiotic - usually conferred by a plasmid carrying the antibiotic resistance gene. ...

...Immunocytochemistry You may also like... Antibody Plasmid Collection Antibody Blog Posts Antibody Guide Western Blot Protocol...Triton X-100 Primary antibody Secondary antibody Deionized water Microscope slide Anti-fade mounting medium...Secondary antibodies must match the host species of the primary antibody. For example, use an anti-mouse ... with a primary antibody against a target protein and a fluorescent secondary antibody. Protocols...Protocol Recombinant Antibody Purification Protocol Introduction Immunocytochemistry is a technique that...that uses antibodies to detect antigens in cells. Here we describe the basic steps for fixing and labeling... with a primary antibody against a target protein and a fluorescent secondary antibody. This protocol ...

...the primary antibody. For example, use an anti-mouse secondary antibody for primary antibodies raised in...Blot You may also like... Antibody Plasmid Collection Antibody Blog Posts Antibody Guide Immunocytochemistry...Immunocytochemistry Protocol Recombinant Antibody Purification Protocol Introduction Western blot is a technique used...block, incubate with primary antibody Day 2: Incubate with secondary antibody Video Watch this instructional...Chemiluminescence substrate Plastic wrap Primary antibody Secondary antibody Deionized water Before Starting Refer...information specific to your antibody, such as ideal blocking buffer and optimal antibody concentrations. Consider...Consider titrating your antibody to determine the optimal dose. Secondary antibodies must match the host ...

...mammalian cells to produce antibodies Antibody Purification Purify recombinant antibodies using Protein A or ...Titering Assay for Lentivirus Quantify virus by counting antibiotic resistant colonies Generating Stable ...Video! Immunocytochemistry Use antibodies to detect antigens in cells Antibody Validation Using the Indirect...testing your virus preparations. Antibodies Protocols for common antibody applications. Intro to the Lab...lab Watch the Video! Over-Agar Antibiotic Plating Quickly add antibiotic to a pre-poured plate Watch the...Stain Determine purity and concentration of recombinant antibodies Watch the Video! Western Blot Separate ...indirect ELISA against a purified antigen to demonstrate the expected antibody dose response....

...Ampicillin resistance but is much more important for other antibiotic resistances. Plate some or all of the...containing the correct antibiotic. The resistance gene on your plasmid must match the antibiotic on the plate. .... Transformation of bacteria with plasmids is important not only for studies in bacteria but also because...both a bacterial origin of replication and an antibiotic resistance gene for use as a selectable marker.... Pro-Tips Commercial competent cells range significantly in their transformation efficiency. The lowest...amplification. Higher efficiency cells are more important if you will be transforming with very small amounts...device Reagents LB agar plate (with appropriate antibiotic) LB or SOC media Competent cells DNA you'd like...

...precipitated DNA. Supernatant contains residues, salts, and water. Pour out the supernatant in the sink. Open... other contaminants from the plasmid DNA. It is also advisable to add RNAse to the supernatant after step...Promega sell kits for isolating plasmid DNA in quantities as low as a few micrograms to as much as several...150 ng/μL to several μg/μL. The protocol below is meant to describe the general procedure for purifying ...using a kit, follow the kit's instructions. If you want to perform plasmid purification without using a ...aliquot it into several tubes/bottles. Remove the supernatant and resuspend the bacteria in buffer. Note: This...centrifugation, and remove the plasmid-containing supernatant. Add either ethanol or isopropanol to precipitate...

...concentration of the antibiotic in culture, or remove the antibiotic entirely. If the antibiotic is reduced or...different degrees of cell death upon antibiotic selection. It is important to monitor these cells regularly...dose of antibiotic, which may not be stable at 37 °C. To achieve a stable cell pool, the antibiotic selection...transfection. Some lentiviral vectors deliver mammalian antibiotic resistance (e.g., puromycin, blasticidin), which...stable cell culture after transduction. Performing antibiotic selection on transduced cells enables elimination...the transducibility of the cell line used, this antibiotic selection may be a vital step for obtaining a... Note that not all lentiviral vectors deliver antibiotic resistance. This protocol was established using...

...21bp antisense—TTTTTG 3’ Reverse oligo: 5’ AATTCAAAAA—21bp sense—CTCGAG—21bp antisense 3’ For... literature by Addgene is not meant to carry with it, and does not grant any license or rights of access...of materials by Addgene is not meant to carry with it, and does not grant any license, express or implied...protocol is provided for your convenience. See “warranty information” in appendix. Table of Contents A....Sequence of pLKO.1 TRC-Cloning Vector I.2 Recipes I.3 Warranty information Back to Top A. pLKO.1-TRC Cloning ...for cloning into pLKO.1, insert your sense and antisense sequences from step B.1 into the oligos below....below. Do not change the ends; these bases are important for cloning the oligos into the pLKO.1 TRC-cloning...

...restriction sites are present in a given sequence. You want to choose enzymes that: Do not cut within your insert...Next, we need to examine the DNA sequence that we want to amplify and design primers that will bind to ...primer design: Because we are cloning an ORF, we want to clone from the start codon (ATG) to the stop ...Product Run PCR to amplify your insert DNA. It is important to use a high fidelity taq polymerase to minimize.... The fidelity of the polymerase becomes more important the longer the expected PCR product is. You should...some DNA during the gel purification step, it is important to digest plenty of starting material. We recommend...be cut with both enzymes, and therefore it is important that the digest goes at least 4 hours and as long...

...information on nucleic acid quantification, see our protocol for DNA quantification , which can be modified...more hardy tissues such as bone, or bacteria/yeast/plant samples will require additional steps to effectively...Incubate at -20°C for 1 hour. Pro-Tips If you anticipate your RNA yield to be small, RNase-free Glycogen...minutes at 10,000 x g at 4°C and discard the supernatant. There should be a gel-like white pellet of total...minutes at 10,000 x g at 4 °C and remove the supernatant. Remove as much of the ethanol wash as possible...Air-dry the pellet for 5-10 minutes. Critical It is important to not let the pellet get too dry before resuspending...

...in the recipient plasmid. (You don't want to express the antisense version of your gene!) Are in frame ...sequence. When selecting restriction enzymes, you want to choose enzymes that: Flank your insert, but do...some DNA during the gel purification step, it is important to digest plenty of starting material. We recommend...be cut with both enzymes, and therefore it is important that the digest go at least 4 hours and as long...running a gel for purification purposes it is important to have nice crisp bands and to have space to ... your favorite gel purification method, it is important to determine the concentration of recovered DNA...DNA in a standard ligation reaction. You ideally want a recipient plasmid to insert ratio of approximately...

.... See below for options if the recombinant band makes up a significant proportion of the DNA pool. Last...Prewarm 3X LB Agar + Antibiotic plates at 37 °C. Prewarm 8X LB Agar + Antibiotic Bioassay plates. Prechill...freeze-thaw if not suspended in cryoprotectant like Glycerol or DMSO solutions. Quantify the individual Maxipreps...Amplification is usually necessary to produce sufficient quantities of library for experimental applications. Repeated...bacterial propagation (origin of replication and antibiotic selection). This recombination, at a low rate...efficiency uptake of plasmid library DNA. This quantity of cells is sufficient for libraries up to 200,000...recovery media (Lucigen, 80026-1) 8X LB Agar + Antibiotic 245 mm bioassay plates (Molecular Devices, X602...

...one or more antibiotic resistance genes, which confer resistance to a specific antibiotic to the bacteria...you will usually want to start 2 mL in a falcon tube, but for larger preps you might want to use as much...Isolating Your Plasmid DNA . Antibiotic Concentrations Commonly Used Antibiotics Recommended Concentration...times. Double check that the antibiotic in your LB media matches the antibiotic resistance on your plasmid...bacteria carrying them. The presence of an antibiotic resistance gene on a plasmids allows researchers to.... growing the bacteria in the presence of the antibiotic). Luria broth (LB) is a nutrient-rich media commonly...LB to a tube or flask and add the appropriate antibiotic to the correct concentration ( see table below...

...agarose gels, so you will want to stay in the 0.7-0.8% range if possible. You will want nice crisp bands. This...and running the gel at a lower voltage. You will want to have enough space around each band to cut without...the gel before cutting out the bands and you will want to use long-wavelength UV for as short a time as...available. Unlike the plastic tray, this will not significantly reduce the UV, but will protect the UV box from...around the band as possible. To do so, it is often important to take the excised band, lay it down on the UV...sides with the razor blade. This is especially important during the DNA purification step, as many kits...during the DNA isolation step. Finally, you will want to isolate the DNA from the gel. This is most commonly...