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...construct transiently care needs to be taken to ensure that the proper cell line is used; several common...

...manage a large number of files as part of the job. Structure of a cover letter Cover letters follow the same...

...liquid culture. More aeration may help to increase the density of the culture. Normally cultures shake ... to room temperature. Now screw on the top of the bottle and store the LB at room temperature. When ready...protocol and conduct an OD600 to measure the density of your culture if needed. A good negative control...from the bacterial culture by following the Isolating Your Plasmid DNA protocol. Figure 1: Media without...went wrong? Try growing the culture for more time. Some bacterial cultures grow more slowly. Also, bacteria...Inoculating a Bacterial Culture Protocols... Protocols Inoculating a Liquid Bacterial Culture Inoculating a Liquid Bacterial...

...break down at this temperature. Check with your antibiotic’s manufacturer to make sure this is true in your...grow). However, it is also more expensive. Procedure Measure 37g of pre-mixed LB-agar powder per L of molten... min. The high pressure will prevent your gel mix from boiling over at high temperature. Pro-Tip Although...harsh conditions in the autoclave. Be sure to check the literature for an appropriate sterilization technique...60 ℃ is a good temperature because the molten agar will remain liquid at this temperature but most antibiotics...bottle. Pro-Tips Be sure to swirl your plates after pouring to remove bubbles and ensure an even distrubtion...plates to solidify at room temperature, however we leave them out at room temperature overnight to allow them...

...Plasmid DNA from Bacterial Culture Recovering Plasmid DNA from Bacterial Culture You may also like... Bacterial...will discuss the general procedure for purifying plasmid DNA from bacterial culture. For details on how to...describe the general procedure for purifying plasmid DNA from bacterial cultures. If you will be using...(low copy plasmids require larger cultures). Centrifuge the culture to pellet the bacteria before proceeding...How to purify plasmid DNA from a bacterial culture, including protocols, tips, and FAQ Protocols...Isolating Bacteria Inoculating a Liquid Bacterial Culture Introduction Many molecular biology techniques ...individual colonies and to generate liquid bacterial cultures , please see those pages. Several companies, such...

...individual cultures. Other methods for enhancing single cell growth, such as using culture medium with...different cell types. Before starting this procedure, be sure that the cell pool has been sufficiently ...trypsinize the cells and expand them to larger culture dishes, being sure not to cross contaminate the independent...highest or lowest transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal cell lines...of proviral integration across cells. Selective pressure on this heterogeneous cell pool could lead to ...TrypLE, Thermo Fisher 12605-010) 6-well tissue culture treated dish, Corning 3516 96-well plate, Corning... µm cell strainer mesh (optional) 10 cm tissue culture dish, Corning 430167 (optional) Reagent Preparation...

...walk-in refrigerators to achieve a temperature between 4°C and room temperature. This general protocol suits...bath weights and floats Reagents None needed Procedure Ensure that the water bath and water bath cover are...spot to secure the thermometer in place. Turn the water bath on and set the appropriate temperature as described...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...amount of water needed to fill the water bath, and be sure to know what the maximum fill capacity is. Look ...described in your protocol. How to set the temperature will vary depending on the model of water bath,...

...Grade Water Procedure Preparation Before handling any viruses, get materials ready. Ensure that primers...the cartridge with the DG8 gasket, making sure that it is secure. Transfer the cartridge holder to the droplet...is not at temperature, touch the screen on the plate sealer to allow it to get to temperature. Once the...of the titer, take the average of several measurements. Figure 1: ddPCR sample data Sample Dilution Concentration... BSL-2 handling depending on the insert. Please ensure that you are in compliance with your institution...place in the Biological Safety Cabinet (BSC). Make sure that the BSC to be used for dilution and the BSC... droplet generation (droplet generation BSC) to ensure no contamination of the PCR reagents and the NTC...

...carefully puncture the QuickSeal tube at the interface of the 60% and 40% gradient (see Figure 2) with ...using Iodixanol gradient ultracentrifugation to get pure adeno-associated virus (AAV) preps for use in your...of steps that separate out contaminants from an impure AAV preparation. The 15% iodixanol step has 1M ...of Buffer B and 2 mL of 5 M NaCl to 43 mL PBS Procedure Preparation and loading of the iodixanol gradient... Hamilton needle, taking care to avoid bubbles (Figure 1). 8 mL of 15% iodixanol step 5 mL of 25% iodixanol...the rotor and place them in a stable rack. **Make sure not to disturb the gradient!** Collect Fractions...the needle’s opening to collect the fractions. Puncture the top of the QuickSeal tube with a 16 ga needle...

.... Addgene makes every effort to ensure the accuracy of its literature, but realizes that typographical... any kind regarding the contents of any literature. Literature are provided to you as a guide and on an... pLKO.1 allows for convenient stable selection. Figure 1 : Map of pLKO.1 containing an shRNA insert. The...70°C for 10 minutes then slowly cool to room temperature over the period of several hours. If using a ...the flame, and allow the water to cool to room temperature. This will take a few hours, but it is important...alternative options, please refer to the enzyme manufacturer's resources, such as NEB’s online tool NEBcloner...gel extraction kit. Elute in 30 μL of ddH 2 O. Measure the DNA concentration. C.4 Ligating and Transforming...

...recovery of cells. Ensure access to autoclaved, sterile reagents for all steps. Procedure Day 1 Add 200 ng...Pooled libraries can be challenging and expensive to ensure adequate quality, but this upfront cost will save...Transformation should be performed at the end of the day to ensure that growth times are limited. Day 2: Harvest cells...should be harvested first thing in the morning to ensure that outgrowth and competition are limited. Equipment...The use of electrocompetent cells is essential to ensure high efficiency uptake of plasmid library DNA. ...Repeat for each of the 25 µl aliquots of cell/DNA mixture used (8 in the described protocol) using a new ... plates upside down at 30 ℃ overnight. Critical Ensure at this stage that no unabsorbed media drips onto...

... you can see the edges of the aperture are in focus. Center the aperture in your field of view and then...drawing skills. Regardless of your image capture method, be sure to record the magnification used, any stains...images in the future. Conclusion Like any important laboratory instrument, you should be sure to take care...a drop of pond water or the cells in your cell culture dish. Microscopes come in a huge range of shapes...ability to distinguish between two discrete objects). Figure 1 depicts an image of a compound light microscope...microscope with the main components labeled: Figure 1: Diagram of a compound light microscope with labels. Created... You can increase contrast by a) adjusting the aperture of the condenser diaphragm to limit the amount...

...video content, including how-to screen captures, lab procedure protocols, and career advice videos....Inoculating a Liquid Bacterial Culture Protocol Getting Started with Tissue Culture Tips and tricks for working...working with tissue culture in the lab. Blog post: 10 Basic tips for mammalian cell culture Aseptic Technique...Video protocol guides for standard laboratory procedures. (Link opens in a new window) Video Link Description...Related Page Making LB Agar Plates Create plates to culture bacteria in the lab Pouring LB Agar Plates Protocol... Over-Agar Antibiotic Plating Create plates to culture bacteria in the lab Over-Agar Antibiotic Plating...Transformation Protocol Inoculating a Liquid Bacterial Culture How to prepare and grow bacteria in a liquid medium...

...the cartridge with the DG8 gasket, making sure that it is secure. Transfer the cartridge holder to the droplet...is not at temperature, touch the screen on the plate sealer to allow it to get to temperature. Once the...generally considered biosafety level 2+. Please ensure that you are in compliance with your institution...Purification Kit, Thermo Fisher, K0721 6-well tissue culture treated dish, VWR, 29442-042 DMEM, high glucose...15 mL DMEM Complete 3 µL of 250 U/µL benzonase Procedure Transducing Cells Thaw the virus on ice. Prepare...Gently aspirate media from the 6-well plates. Be sure to use a new aspirating pipette for each well as... it in the Biological Safety Cabinet (BSC). Make sure that the BSC is supplied with sufficient pipette...

...PCR machine. Set annealing temperature 5°C below the primer melting temperature (Tm). Set extension step... See manufacturer’s instructions for specific instructions about extension time and temperatures. Initial...designed to have similar melting temperatures (Tm). Set the annealing temperature to 5°C lower than the Tm of...radical temperature changes during a typical PCR. The DNA polymerase has an optimum temperature around ...withstand many heating and cooling cycles, which would denature DNA polymerases from other species. In addition...denaturation step is commonly performed at 94–98°C. Denature 30 seconds at 94°C: Continued denaturation of ...forward and reverse primers are stable within this temperature range to anneal to each of the single stranded...

...puncturing your glove. Ensure that the tip is properly set on the pipette to ensure pipetting accuracy. ...working in a laboratory, properly dispensing liquid ensures the accuracy of your experiment and any changes...Pipette tips Waste container Containers to hold measured liquid (ex: microfuge tube, bottle, etc.) Labels...read as 100 from top to bottom (as shown in the picture above), while 650 µL would read as 065. P200: The...read as 100 from top to bottom (as shown in the picture above), while 95 µL would read as 095. P20: The...Therefore, 10 µL would read as 100 (as shown in the picture above), while 2.2 µL would read as 022. P2: The...Therefore, 1 µL would read as 100 (as shown in the picture above), while 0.5 µL would read as 050. Each volume...

...electrophoresis chamber and secure it. Note: Check the manufacturer’s instructions if you are unsure of the correct...follows: Figure 2 Using the box tool, draw a box around the entire first gel lane (as in Figure 2). Select...with Coomassie blue, protein band intensities are measured using ImageJ software and a standard curve is ... mL of deionized water. Gently invert to mix. Procedure Section 1: SDS-PAGE Add 5 µL of 4X sample buffer...Fill the chamber with 1X MOPS running buffer. Make sure that the chamber is sealed. Load 5 µL of the prestained... of the gel with an appropriate imaging system. Figure 1 Recombinant antibody preps should have 2 clear...electrophoretic mobility of the bands (sample AR0016 in Figure 1) indicates that the samples may not have been...

... web-based primer design software to ensure a melting temperature between 50-60°C for your PCR primers...room temperature, then add 1 μl of 25 mM EDTA, followed by another 5 minutes at room temperature. The ...separation of linearized vector from the reaction mixture, and may provide counter-selection for negative...negative clones as in the example shown here. The manufacturer of an LIC-specific vector will provide the homologous...LIC is often as simple as using the backbone manufacturer's suggested leader sequence fused to your gene...separate the linearized vector from the reaction mixture by gel electrophoresis followed by gel purification...products with sterile water (instead of TE buffer) to ensure optimal salt concentrations in subsequent reactions...

...These lab safety guidelines provide steps to ensure you are working in BSL-1 and BSL-2 labs safely....different types of hazards and to provide protective measures for each level, as each level has different safety...pathogens including organisms such as Staphylococcus aureus or Vibrio cholerae . BSL-2 includes all of the ...precautions to prevent injuries, ingestion, and exposures to hazardous materials. Video Watch the video ...laboratory. Before you start your experiment, make sure your workspace is clean and uncluttered. Disinfect...the appropriate contact times for each agent. Make sure you have enough space to work at your lab bench....are also required to be present in the room. Make sure that you know where these are located before you...

... some cells in the culture may adversely affect the surviving cells in the culture, so it is important...blasticidin), which enables selection of a stable cell culture after transduction. Performing antibiotic selection...Microcentrifuge tubes, Neptune 3745.X 6-well tissue culture treated dish, Corning 3516 15 mL conical tubes,... be subjected to multiple freeze-thaw cycles. Procedure Before beginning, determine the optimal dose of... mL of virus solutions at various dilution. Make sure to use the polybrene-containing media to make the...stable cell pool. Observe the dish every day to ensure that the cells in the untransduced well (0 µL lentivirus... reduce the concentration of the antibiotic in culture, or remove the antibiotic entirely. If the antibiotic...

...buffer (see manufacturer's instructions) Gel loading dye Electrophoresis buffer Pipet tips Procedure Select...at appropriate temperature (usually 37 °C) for 1 hour. Always follow the manufacturer’s instructions. .... Follow the manufacturer’s instructions. If your enzyme did not cut, check to make sure that it isn't... like... Recovering Plasmid DNA from Bacterial Culture Purifying DNA from an Agarose Gel DNA Ligation ...amounts) Appropriate restriction enzyme (see manufacturer's instructions for proper ammount) Approrpriate...Restriction Enzyme(s) Buffer BSA (if recommended by manufacturer) dH 2 O up to total volume Pro-Tips The amount...window) QIAGEN DNA cleanup kit . See the enzyme manufacturer's instructions for more details. To visualize...

... at -20 °C Procedure Section 1: Affinity chromatography Equilibrate filtered tissue culture supernatant...affinity purify recombinant antibodies from cell culture supernatant using Protein A or Protein G columns... following your institution’s hazardous waste procedures. Last Update: February 7, 2022 Protocol Video..., 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...part Protein A/G binding buffer to 1 part tissue culture supernatant and mix well. Uncap the Protein G Gravitrap...

...glycerol stocks. Procedure Follow the steps for Inoculating an Overnight Liquid Culture . After you have... appropriate temperature. Growth conditions, including copy number and growth temperature, can be found...you retrieve your liquid bacterial culture, take 500 μL of culture to make your glycerol stock before ...times). Make sure that you see one uniform solution, and there are no layers present. Be sure to label both...Transformation Recovering Plasmid DNA from Bacterial Culture Background Information Bacterial glycerol stocks... bacterial growth, add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw top tube...next day you will be able to start an overnight culture for plasmid DNA prep the following day. Tips and...

...cm tissue culture plates. Incubate the cells at 37 °C, 5% CO 2 for ~20 h. Prepare a mixture of the three... 113.4 Gently add the diluted PEI mixture to the diluted DNA mixture. Add the diluted PEI dropwise while...polyethylenimine (PEI) transfection protocol. This procedure can be modified for alternative packaging cell...Microcentrifuge tubes, Neptune 3745.X 10 cm tissue culture dish, Corning 430167 15 mL conical tubes, VWR 21008...solution to mix for 10 min and then recheck the pH to ensure that it has not drifted. Filter the solution through...that are below passage 15 for viral production. Procedure Seed 293T packaging cells at 3.8×10 6 cells per...negative E. coli strain such as NEB stable. Make a mixture of a total of 500 μL PEI-OptiPro SFM with enough...

... counter and measure the live cell density and viability of the culture. Pro-Tip Culture should be between...20 °C. Procedure Section 1: Seeding cells The day prior to transfecting, seed a 108 mL culture of HEK293...transient expression of a gene of interest in cell culture. This protocol describes how to transfect suspension... cells are considered biosafety level 2. Please ensure that you are in compliance with your institution...the DNA and working stock of PEI-MAX to room temperature before use, and warm the BalanCD HEK293 transfection...water to a final volume of 1 L and recheck pH to ensure that it has not drifted. Filter the solution through...biosafety cabinet and incubate for 1 h at room temperature. After the BCD TFX has warmed in the bead bath...

...samples at specific temperatures. This protocol will cover the general procedure and features to keep in mind...densities is essential to many different experimental procedures, such as minipreps and RNA extractions. Plus,... centrifuge may have specific instructions so be sure to check in with someone familiar with your lab’...the time and speed needed for your experiment. Procedure An example of a balanced tabletop centrifuge, ...a tabletop microcentrifuge like the one in the picture above. Wear PPE appropriate for the lab space in.... Before using the centrifuge for your samples, ensure that the centrifuge is clean and that everything...center (see image to right). Even distribution ensures that the centrifuge is balanced. Using the centrifuge...

...Biosafety Levels One and Two (BSL-1 and BSL-2) Safety measures for laboratories operating at BSL-1 and BSL-2 ...the building blocks for many more complicated procedures. Name Description (Link opens in a new window...to Video Making LB Agar Plates Create plates to culture bacteria in the lab Watch the Video! Over-Agar ...Antibiotic Plating Quickly add antibiotic to a pre-poured plate Watch the Video! Streaking Bacteria Isolate...Watch the Video! Inoculating a Liquid Bacterial Culture Prepare and grow bacteria in liquid medium Watch...extract, and precipitate DNA DNA Quantification Measure DNA concentration with a spectrophotometer Restriction...Video! Sequence Analysis Verify important plasmid features using sequence analysis RNA Extraction Without...

...and click measure, be sure to record the concentration and purity. Note: Purity is measured under the ...Inoculating a Liquid Bacterial Culture Recovering Plasmid DNA from Bacterial Culture Restriction Digest of Plasmid...substance in that liquid. In order to accurately measure the concentration of a substance based on its absorbance... of whether you have a NanoDrop, follow the manufacturer's instructions for the spectrophotometer specific...Spectrophotometer Tips Before measuring any samples, be sure to ‘blank’ the spectrophotometer using the solution...resuspended in, but with no DNA added. 'Blanking' measures the background inherent to the machine and your...your solvent. If using a NanoDrop to measure your samples, place 1-2µL of mini-prepped DNA onto the pedestal...

...list above. Procedure Option #1 - Solution D Protocol Before starting this protocol, make sure to prepare...TRIzol® User Guide from ThermoFisher Scientific . Figure 1: A diagram of the different steps in RNA extraction...step, Option B) Glycogen (Optional) Caution Make sure to read the SDS (Safety Data Sheet) for safety warnings...use 1 mL of Solution D per 100 mg of cells. For cultured cells: use 1 mL of Solution D per 1 X 10 7 cells...cells. Allow sample(s) to sit at room temperature for 5 minutes to allow for dissociation of the nucleoprotein...centrifuge, bring this equipment back to room temperature, as prolonged storage in the cold room may damage...top, aqueous phase to a new RNAse-free tube. The mixture separates into a bottom organic layer, an interphase...

...DNA from Bacterial Culture Introduction If you have a glycerol stock or stab culture of bacteria and need...inoculate a bacterial culture for DNA purification will minimize the chance of having a mixture of plasmids in...touch the bacteria growing within the punctured area of the stab culture or the top of the glycerol stock.... (with appropriate antibiotic) Bacterial stab Procedure Obtain an LB agar plate with appropriate antibiotic...sterilize it by passing it through a flame, just be sure to allow enough time for the loop to cool before...diagonal lines in another section of the plate. Make sure that the first line (and only the first) in each...plasmids, which sometimes recombine at 37 °C. Be sure to check this before incubating your plate. In the...