... Protocols General Transfection General Transfection You may also like... Viral...Service Introduction This protocol describes a general method for transfecting mammalian cells using linear...discard the tube and thaw a new working stock. Considerations Before You Start The health of the cell line...

... Taq polymerase has an optimal temperature around 70-75°C so this step enables the DNA polymerase to synthesize...synthesis. Taq DNA polymerase: Special DNA polymerase that can withstand radical temperature changes during...resistant DNA polymerase from the thermophilic bacterium, Thermus aquaticus (Taq) . Taq polymerase can withstand...denature DNA polymerases from other species. In addition to the template DNA and the Taq polymerase, PCR requires...creates several micrograms of target DNA from just a few nanograms of template DNA through several cycles...PCR machine. Set annealing temperature 5°C below the primer melting temperature (Tm). Set extension step...designed to have similar melting temperatures (Tm). Set the annealing temperature to 5°C lower than the Tm of...

... Protocols Generating Stable Cell Lines with Lentivirus Generating Stable Cell Lines with Lentivirus...Protocol to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector...Service Introduction This protocol can be used to generate stable cell lines expressing a gene of interest...observed using transient transfection approaches, generating cell lines using lentiviral vectors enables long-term... in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health of the target cell...polyclonal populations of resistant cells start proliferating and the individual wells become confluent, expand... over the culture. To overcome this, consider generating monoclonal lines from the early polyclonal populations...

... master mix preparation and droplet generation (droplet generation BSC) to ensure no contamination of ...cap the PCR tubes. Generate the Droplets Bring the PCR tubes to the droplet generation BSC. Without disturbing...holder to the droplet generator. Close the lid and wait for the droplets to be generated. Once the droplets...is not at temperature, touch the screen on the plate sealer to allow it to get to temperature. Once the...increase the accuracy of the titer, take the average of several measurements. Figure 1: ddPCR sample data...12–5E+13 GC/mL, where GC is genome copies and generally represents the physical number of viral particles...surfaces with 10% bleach. Safety Warnings AAV is generally considered biosafety level 1 but may require BSL...

...for generating stable cell lines with lentivirus . Day 0: (optional) Seed cells for the generation of ...theory a monoclonal line could be generated at any time. Considerations Before You Start Some cell lines...number of monoclonal lines generated using this protocol will vary based on several factors, including the...seeding the plate at an average density of 0.5 cells/well. Seeding an average of 0.5 cells/well ensures...This protocol describes how to generate a monoclonal cell line from a polyclonal pool of stable cells...Service Introduction This protocol describes how to generate a monoclonal cell line from a polyclonal pool ...expressing clones take over the polyclonal cell pool. Generating a monoclonal cell line by limiting dilution results...

...science. General Considerations The ELISA can be completed in a single day or broken up over several days ...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...incubating certain steps at 4 °C instead of room temperature or 37 °C. The protocol notes when there are different...1L Foil Before Starting Warm reagents to room temperature. Procedure Section 1: Prepare the Antigen Standard...into 999.5 mL 1X PBS Cap the bottle and invert several times to mix. Carefully remove the plate seal from...of BSA to 25 mL of PBS. Cap the tube and invert several times to mix. Using a multichannel pipette, add...microplate shaker set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section 3: Primary antibody...

...holder to the droplet generator. Close the lid and wait for the droplets to be generated. Once the droplets...is not at temperature, touch the screen on the plate sealer to allow it to get to temperature. Once the...increase the accuracy of the titer, calculate an average of several dilutions. Figure 1: ddPCR Lentivirus sample...1E+09 TU/mL, where TU is transducing units and generally represents the number of infectious viral particles...with 10% bleach. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please ensure that...Supermix for Probes no dUTP, Bio-Rad,1863023 Droplet generation oil, Bio-Rad, 1863005 DG8 cartridge, Bio-Rad,...tubes. Pipette back and forth 10 times to mix. Generating the Droplets Place a DG8 cartridge into the cartridge...

...ligation. Because of its dual polymerase/exonuclease functions, T4 DNA polymerase can create overhangs of varying... Because the polymerase reaction is favored over the exonuclease reaction, the polymerase will add back...room temperature, then add 1 μl of 25 mM EDTA, followed by another 5 minutes at room temperature. The ... of the enzyme and shift its activity back to polymerase. In our case, dGTP will be used in the reaction...primers will have different leader sequences, but operate on the same principle (homologous to the first ...web-based primer design software to ensure a melting temperature between 50-60°C for your PCR primers. Step 2:...Overhangs Treat the linearized vector with T4 DNA polymerase to "chew back" the free 3' ends, following the...

...Human U6 promoter drives RNA Polymerase III transcription for generation of shRNA transcripts. cPPT Central...minutes then slowly cool to room temperature over the period of several hours. If using a beaker of water... any kind regarding the contents of any literature. Literature are provided to you as a guide and on an...complex in the transduced cells. hPGK Human phosphoglycerate kinase promoter drives expression of puromycin...information, visit Addgene’s Mammalian RNAi Tools page. Several other laboratories have deposited pLKO derived ...because polyT is a termination signal for RNA polymerase III. Note that these were the prevailing guidelines... B.2 Ordering Oligos Compatible with pLKO.1 To generate oligos for cloning into pLKO.1, insert your sense...

... colonies and to generate liquid bacterial cultures , please see those pages. Several companies, such ...to as much as several miligrams and at concentrations ranging from 150 ng/μL to several μg/μL. The protocol...good idea to let it sit for several hours to overnight at room temperature before quantifying and using...concentrated plasmid DNA. This page will discuss the general procedure for purifying plasmid DNA from bacterial...protocol below is meant to describe the general procedure for purifying plasmid DNA from bacterial cultures... TE buffer 3 M Na-acetate (pH 4.8) Protocol: Generalized DNA Purification Grow an overnight culture of...into a single centrifuge tube, aliquot it into several tubes/bottles. Remove the supernatant and resuspend...

...walk-in refrigerators to achieve a temperature between 4°C and room temperature. This general protocol...the appropriate temperature as described in your protocol. How to set the temperature will vary depending...the correct temperature. Set up your water bath so that it will reach the desired temperature by the time...that helps bring your materials to a particular temperature, catalyze chemical reactions such as restriction...prevent evaporation and maintain the desired temperature. This also helps the water bath heat up quicker... hour before you need to use it to allow the temperature to stabilize. Use water resistant markers, such... them. Once the water bath is at the correct temperature, remove the lid and place your tubes or bottles...

... boiling over at high temperature. Pro-Tip Although our recommended temperature should kill most potential...60 ℃ is a good temperature because the molten agar will remain liquid at this temperature but most antibiotics...plates to solidify at room temperature, however we leave them out at room temperature overnight to allow them...conditions in the autoclave. Be sure to check the literature for an appropriate sterilization technique if...antibiotics will not break down at this temperature. Check with your antibiotic’s manufacturer to make sure... to. To be certain your agar is at the right temperature, we recommend using a laser thermometer. Light...both plates overnight at the appropriate growth temperature and check for growth. See our sample data section...

... high fidelity taq polymerase to minimize mutations. The fidelity of the polymerase becomes more important...You should select an annealing temperature based on the melting temperature (Tm) of the portion of the primer...bp depending on the polymerase used. Because of this, no matter which taq polymerase you use, it is important... site to improve cutting efficiency. You can generally add any 6 bases, but you should ensure that the... 18bp of homology to the ORF). We now need to generate the reverse-complement of this sequence so that...that we can successfully amplify the ORF. You can generate the reverse-complement using existing software...you can use a pretty wide range of annealing temperatures, but you may need to increase your primer length...

... to support reproducibility in science. General Considerations For antibody-expressing plasmids containing...Antibodies Blog Posts Molecular Biology Reference General Transfection Protocol Recombinant Antibody Purification...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...Harvest antibody Equipment Biosafety Cabinet 4 °C Refrigerator Micropipettes and tips Pipettor and pipettes...the DNA and working stock of PEI-MAX to room temperature before use, and warm the BalanCD HEK293 transfection...until all particles have dissolved. This may take several hours. Adjust to pH 7.0 with 10 N sodium hydroxide...biosafety cabinet and incubate for 1 h at room temperature. After the BCD TFX has warmed in the bead bath...

...1 and BSL-2) Safety measures for laboratories operating at BSL-1 and BSL-2 Watch the Video! Water Baths...plasmid using restriction enzymes Watch the Video! Polymerase Chain Reaction (PCR) Basic PCR protocol with ...Watch the Video! How to Design a Primer Key considerations when designing primers Watch the Video! Sequence...restriction digest and ligation Cloning by PCR Generate restriction sites by PCR Modification by Annealed...cloning with Type II restriction enzymes and T4 polymerase pLKO.1 - TRC Cloning Vector Cloning protocols...mouse genes. Virus Name Description Link to Video General Transfection Introduce plasmid DNA to mammalian...virus by counting antibiotic resistant colonies Generating Stable Cell Lines with Lentivirus Genomically...

...10, 2020 Equipment Refrigerated microcentrifuge If you only have a non-refrigerated microcentrifuge, see...extract RNA from tissues or cells without a kit. The general steps of this protocol include homogenization/lysis...X 10 7 cells. Allow sample(s) to sit at room temperature for 5 minutes to allow for dissociation of the...RNA degradation. If you don’t have access to a refrigerated centrifuge, you can carefully bring a centrifuge...centrifuge, bring this equipment back to room temperature, as prolonged storage in the cold room may damage...

... keep samples at specific temperatures. This protocol will cover the general procedure and features to...achieves this by using centrifugal force, which is generated by spinning the sample at a high speed. Being ...the bottom of a tube. You will likely encounter several different types of centrifuges that can accommodate...almost any centrifuge. Types and Parts There are several types of centrifuges: Mini or Tabletop Centrifuges... larger containers. Ultracentrifuges are also generally floor sized centrifuges that can hold larger containers...centrifuge settings according to speed and time (and temperature, if applicable) listed in your protocol. Pro-...

...capabilities. Taking into consideration the information above, primers should generally have the following properties...Primers How to Design a Primer You may also like... Polymerase Chain Reaction Plasmid Cloning by PCR Agarose... 5’ end of the primer usually has stretches of several nucleotides. Also, both of the 3’ ends of the hybridized...primers result in a slower hybridizing rate. On average, the DNA fragment that needs to be amplified should...content Start and end with 1-2 G/C pairs Melting temperature (Tm) of 50-60°C Primer pairs should have a Tm...

...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...Class II, Type A2 Biological Safety Cabinet 4 °C Refrigerator Pipette controller Benchtop centrifuge compatible..., binding buffer, and elution buffer to room temperature (RT) before use. Reagent Preparation 1 M sodium... containing the recombinant antibody to room temperature (see Transfection for Recombinant Antibody protocol...30 kDa MWCO buffer exchange column. (Optional) Regenerate the column by washing with 25 mL of Protein A...remaining space in the column with PBS and pipette several times to mix. Centrifuge at 3100 x g for 8 min ...

...amplification. This protocol is designed to be as general as possible but note that individual libraries ...our protocol on Bacterial Transformation . The generalized pooled library amplification protocol is expected... a diagnostic digest and high-throughput next generation sequencing (NGS). Select a restriction enzyme...10 colonies on the most diluted plate for 1000x coverage of a library of 100,000 plasmids. Colonies may... require extra incubation time in order to be enumerated accurately. Frequently the number of colonies...Ideally, dilutions would have been sufficient to enumerate single colonies but as long as one can ensure ...upon intended use. Typically yields range from several hundred micrograms to a milligram of total plasmid...