... of cold 4% paraformaldehyde in PBS on ice for 15 min . Pro-Tip While 4% Paraformaldehyde fixation works...labeling HeLa cells for a target protein using the formaldehyde fixation method. The protocol may need to be...coated coverslips HeLa cells 24-well plate 4% Paraformaldehyde 5 mg/mL 4′,6-diamidino-2-phenylindole (DAPI...be better for some applications. Remove the paraformaldehyde and follow your institution's laboratory safety...concentration in antibody dilution buffer. Pro-Tip The optimal antibody concentration will vary but generally ...fluorescent filters. Tips and Troubleshooting The optimal fixation method will vary depending on the sample...

...Web resources Addgene’s mammalian RNAi website: www.addgene.org/mammalianrnai The RNAi Consortium (TRC...Designing shRNA Oligos for pLKO.1 B.1 Determine the optimal 21-mer targets in your gene B.2 Order oligos compatible... F.1 Recommended materials F.2 Determining the optimal puromycin concentration F.3 Protocol for lentiviral...resistance gene for selection of pLKO.1 plasmid in mammalian cells. sin 3’LTR 3’ Self-inactivating long terminal...’s lab. For more information, visit Addgene’s Mammalian RNAi Tools page. Several other laboratories have...Designing shRNA Oligos for pLKO.1 B.1 Determining the Optimal 21-mer Targets in your Gene Selection of suitable...Weinberg lab. For more information, visit Addgene’s Mammalian RNAi Tools page. E.2 Protocol for Producing Lentiviral...

...represents the first decimal place. For the P20, this represents the second decimal place, and for the ...pipette in many scenarios to accurately dispense small amounts of liquid (think: 0.1 µL to 1 mL). When ...volume display on these pipettes will also have small tick marks at the bottom. For the P1000, this represents...the P2 this represents the third decimal place. Procedure How to Pipette Now that you know the different...pipetting from a relatively large container holding a small volume of liquid, tilt the container holding the...accurately and precisely is important since even small changes in the volume could affect your experiments...

...buffer and optimal antibody concentrations. Consider titrating your antibody to determine the optimal dose.... concentration in blocking buffer. Pro-Tip The optimal concentration will vary between antibodies but ...detect the bands. Tips and Troubleshooting The optimal lysis buffer will vary depending on the sample ... buffers to find the best for your target. The optimal concentration and blocking buffer will vary between...consider titrating your antibody to determine the optimal dose. To ensure that your antibody is both functioning...

...overlapping DNA fragments can be joined in a single isothermal reaction. With the activities of three different...two-part Gibson reaction if you're only making a small change in a plasmid (such as point mutations). Generate...concentrations. The Gibson Assembly master mix (isothermal reaction mix) consists of three different enzymes...-Stranded DNA-Binding protein (ET SSB) to the isothermal reaction mix. ET SSB protects 3’ ssDNA ends from... in the Gibson reaction are different than with normal PCR primers (Gibson et al. , 2010). Number of fragments...Cepko C. (2020). A Simple Enhancement for Gibson Isothermal Assembly. bioRxiv . https://doi.org/10.1101/2020.06.14.150979...

...your substance maximally absorbs. In the case of nucleic acid (DNA and RNA), the maximal absorbance is ...and most have a specific wavelength that they maximally absorb. By measuring the absorbance of a liquid...is at 260nm. Protein maximally absorbs at 280nm and the ratio of nucleic acid to protein (260/280) is generally...days, many labs have a NanoDrop, which is a very small spectrophotometer that can accurately read DNA concentration...

...While simple homogenization is effective for most mammalian tissues; more hardy tissues such as bone, or bacteria...Pro-Tips If you anticipate your RNA yield to be small, RNase-free Glycogen may be used as a carrier to...cycles of your entire RNA sample, consider making smaller aliquots of it and storing those in -80°C. Option...cycles of your entire RNA sample, consider making smaller aliquots of it and storing those in -80°C. Solution...

...pressure. These recombined plasmids are much smaller and are a smaller nutritional burden on the bacteria. Bacteria...modifications dictated by the originating laboratory for optimal results. If you obtained the pooled library from...can recombine between their LTRs resulting in a smaller plasmid containing only the elements required for...convenience but any brand or house- made LB that supports normal growth is expected to work. 50 mL Falcon Conical...library of 100,000 plasmids. Colonies may appear small and require extra incubation time in order to be...

...through the use of a selection curve to determine optimal antibiotic concentration. Last Update: Oct. 27,...pipetting and spreading Bunsen burner (or other small flame source) Incubator Reagents 6 cm diameter LB... the liquid is mostly absorbed (there is a very small visible volume of pooled liquid remaining on the...over-agar Plate shows several individual colonies with smaller size than the 1 mg/mL and 2 mg/mL plates and effective...

... nearly all plasmids (even those designed for mammalian cell expression) carry both a bacterial origin... cells, which come frozen and are prepared for optimal transformation efficiencies upon thawing. For the...important if you will be transforming with very small amounts of DNA or if you're multiple plasmids at...the cells by centrifugation and resuspend in a smaller volume of LB so that there isn't too much liquid...

...for 1 minute at 72°C: The Taq polymerase has an optimal temperature around 70-75°C so this step enables...creating a “master mix,” which minimizes the number of small volumes you need to pipet. If you are using the ...needs to reconstitute their primers in liquid, normally sterile dH 2 O. To make a 100uM stock of any primer...efficiently. Taq Buffer with MgCl 2 : Provides an optimal and stable chemical environment for the DNA polymerase...

...kDa subunit (RPP30) are used as a control for normalization. The dilution series outlined in this protocol...5425R Droplet digital PCR System, Bio-Rad, DX200 Thermal Cycler, Bio-Rad, T100 PCR Plate Sealer, Bio-Rad...plate has been sealed, proceed to thermocycling. Thermal Cycling Run the following PCR parameters: Cycling...risk of contaminating reagents we recommend making small aliquots of master mixes, and primers/probe mixes...

...Deletions with CRISPR Generate genomic deletions in mammalian cells lines using CRISPR/Cas9 Genomic Deletions...adeno-associated virus from a preparation produced in mammalian cells AAV Purification by Iodixanol Gradient Ultracentrifugation...culture in the lab. Blog post: 10 Basic tips for mammalian cell culture Aseptic Technique Aseptic technique...

...General Transfection Introduce plasmid DNA to mammalian cells Lentivirus Production Produce lentivirus...adeno-associated virus from a preparation produced in mammalian cells Watch the Video! AAV Titration by qPCR Using...to Video Transfection Introduce plasmid DNA to mammalian cells to produce antibodies Antibody Purification...

...Use this protocol to transfect mammalian cells with your plasmid of interest....protocol describes a general method for transfecting mammalian cells using linear polyethylenimine. Transfections...are below passage 20 for viral production. The optimal mass DNA:mass PEI ratio will need to be empirically...

...These tools allow you to observe specimens much smaller than you would be able to with your naked eye, ...advantage of the physical properties of light to detect small objects. Two of the most important properties of...needed, you can also use the fine focus knob (the smaller of the focus knobs) to make minor adjustments to...

...). You might need to express YGOI in cultured mammalian cells. The problem is that the only version of...Using subcloning, you can easily move YGOI into a mammalian expression vector. Design (Choosing enzymes) Many...purpose. Congratulations, you now have YGOI in a mammalian expression vector and can begin your studies. ...

...transfection. Some lentiviral vectors deliver mammalian antibiotic resistance (e.g., puromycin, blasticidin...cycles. Procedure Before beginning, determine the optimal dose of selective reagent for your target cell ...important to do regular media changes and maintain optimal growth conditions for the surviving cells. Even...

...internal passive reference (typically ROX dye), to normalize non-PCR–related fluorescence fluctuations and ... a validated standard curve is obtained, make a small aliquot of each standard (enough for 1 or 2 plates...acceptable) Baseline removal: all samples will have some small amount of background signal that is most evident...

...medium with increased serum concentrations, may be optimal for different cell types. Before starting this ...because the buildup of waste products could be suboptimal for cell growth. This conditioned medium will...10 5 cells/mL) = 0.125 µL Because this is such a small volume, first make 1 mL of a 1:100 dilution of the...