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...tagging of dCas9 can be achieved by fusing a biotin acceptor site to dCas9 and co-expressing BirA biotin ligase...

...of this tutorial we will discuss how to add new restriction sites to the MCS of an empty vector. However...purify vector While waiting for your oligos to arrive, conduct a restriction digest of 1μg of vector with...Mix the annealed oligos with cut vector in molar ratios (vector:insert) between 4:3 and 1:6 in a standard...insert of 50bp in length into a 5kb vector, mix 100ng of the vector with 0.75-6 ng of annealed oligos)....just as easily to add short tags or shRNAs to any vector (the procedure will simply differ in terms of primer...primer design). Let's assume that your favorite vector has a relatively limited MCS (BamHI - EcoRI - SalI...restriction sites and add them to your existing vector. It is a couple days of work that will pay off ...

...annealed but nicked vector product is then repaired during the replication cycle. Empty vectors for LIC typically...your PCR primers. Step 2: Linearize Vector In this example, the vector is cut at two points by the enzyme...subsequent reactions. Step 3: Create Vector Overhangs Treat the linearized vector with T4 DNA polymerase to "chew...treated vector and insert at a molar ratio of 1:2 or 1:3, using between 20 and 50 ng of vector per annealing...allows for electrophoretic separation of linearized vector from the reaction mixture, and may provide counter-selection...shown here. The manufacturer of an LIC-specific vector will provide the homologous sequence which must...Search Addgene's collection of empty LIC cloning vectors Protocol Step 1: Design Your Primers Primer design...

...some of our other resources to help you choose, store, or use your antibody. Antibody Blog Posts Recipient...

... Depositor Collections Bikard Lab - CRISPR Repression Collection...

...colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created...

...into sterile tubes. Store the tubes at -80 °C. After thawing, the solution can be stored at 4 °C for up to...before putting the CS5 in the incubator. Return the CS5 to the incubator for 96 h. This incubation time...Cells You may also like... AAV Viral Service Viral Vector Guides Virus Blog Posts Molecular Biology Reference...Hemocytometer or cell counter Ice bucket CO 2 incubator Pipet controller Hazardous waste container pH ...pH meter Stir plate Magnetic stir bar Sonicator Ear protection Vortex Reagents Adherent HEK293T cells (... glutaGRO 11 mL of 200 mM L-alanyl-L-glutamine. Store at 4 °C. D1 + 0.1 M sorbitol (optional) : DMEM ... solution may separate into phases. Aliquot and store at 4 °C. Cell Lysis Buffer : 50 mM Tris HCl, 150...

... PPE protocols . Working in a BSL-2 laboratory requires laboratory glasses in addition to coat and gloves...also adhered to. None of the authors, contributors, administrators, or anyone else associated with Addgene... eat, drink, chew gum, or apply makeup in the laboratory. Before you start your experiment, make sure ...Only mechanical pipetting should be done in the laboratory. Don’t mouth pipette! There may be some times...officer. BSL-2 Guidelines Remember, the BSL-1 laboratory guidelines above are expected to be followed ...not be worn outside of the BSL-2 area. BSL-2 laboratories must be clearly marked as “BSL-2.” The names...names and contact information of the laboratory manager should be clearly visible in the room and on the ...

...personal protective equipment including laboratory coat, laboratory goggles, and gloves. Sodium azide containing...Class II, Type A2 Biological Safety Cabinet 4 °C Refrigerator Pipette controller Benchtop centrifuge compatible...tubes NanoDrop spectrophotometer 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 °C bead... sterile sodium phosphate dibasic (NaH 2 PO 4 ) Store up to 1 month at 4 °C Adjust pH to 7.0 Autoclave... Autoclave or filter sterilize 1000X protease inhibitor cocktail 25 mg leupeptin 50 mg antipain 250 mg...Recombinant Antibody protocol ). Pro-Tip Add protease inhibitors to 1X if this has not already been done. We typically...of supernatant and add 250 µL of 100X protease inhibitor cocktail. Add 1 part Protein A/G binding buffer...

... are important for long-term storage of plasmids. Although you can store your plasmid DNA at -20°C, many...LB agar plate can be stored at 4°C for a few weeks. However, if you want to store bacteria for a longer...concentration of long-term glycerol storage is unknown. Most labs store bacteria in 15-25% glycerol. You ...for long-term storage. Protocols... Protocols Creating Bacterial Glycerol Stocks for Long-term Storage of Plasmids Creating...Creating Bacterial Glycerol Stocks for Long-term Storage of Plasmids You may also like... Bacterial Transformation...cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years. Protocol Video ...