...: Amplify Insert by PCR Perform PCR amplification of your insert following the instructions provided by...Set up a vector only control with water instead of the insert. Incubate the annealing reaction for 5 minutes...digestion and PCR products with sterile water (instead of TE buffer) to ensure optimal salt concentrations...the free 3' ends, following the manufacturer's instructions. In this case, we will include the free nucleotide...accomplished by gel purification . Step 5: Create Insert Overhangs Treat your purified PCR product with ...Anneal and Transform Mix your treated vector and insert at a molar ratio of 1:2 or 1:3, using between 20...concentration fixed across multiple concentrations of insert, thereby increasing your chances of success. Set...

...use. Negative Result 1: Both Strains Grow Assuming the appropriate strains were streaked on the appropriate...your strains are no longer viable. You can check for this possibility by streaking out both strains on ...slightly. Even so, you should always use thermally insulated gloves when removing anything from the autoclave...appropriate plates, then if both strains grow, it’s possible that: The antibiotic broke down. You forgot to...concentration for selection. Negative Result 2: Neither Strains Grows If neither strain grows, it's possible that...ve used the wrong antibiotic, or confused your strains....

...important plasmid features (such as the gene/insert, fusion proteins, point mutations, deletions, etc.) involves...backbone and can help you verify the ends of the insert. Longer genes may require internal primers that...that anneal to a specific region of the insert. You can find Addgene's sequencing results by clicking on... to verify tags, mutations and a portion of the insert, but we do not sequence the entire plasmid. Addgene...and make sure they are justifiable peaks. For instance, in the trace file below, you can see that just...Sometimes an “N” is the result of an erroneous insertion by the sequencing reaction....

...staining cells in culture with a primary antibody against a target protein and a fluorescent secondary antibody...labeling cells in culture with a primary antibody against a target protein and a fluorescent secondary antibody...need to be optimized for different cells, target proteins, etc. Sharing speeds science. We believe that ... Before Starting Refer to the manufacturer's instructions for additional information specific to your ...Paraformaldehyde fixation works well for many target proteins, it may not be the best fixation method for all...Remove the paraformaldehyde and follow your institution's laboratory safety guidelines for disposing of...between antibodies. Review the manufacturer's instructions before starting your experiment and consider...

...1 or later: Image analysis Video Watch this instructional video to learn how to determine purity and concentration...microcentrifuge. Remove the gel from the plastic wrapper and rinse with deionized water. Gently remove the white sticker...and secure it. Note: Check the manufacturer’s instructions if you are unsure of the correct orientation...the gel. Carefully remove the comb from the gel. Rinse each well with 200 µL 1X MOPS running buffer. Fill... a plastic tray with 100 mL of deionized water. Rinse gel with deionized water for 5 min with gentle agitation...corresponding to the heavy chain (HC) and light chain (LC) proteins, respectively. There should be very little background...import. Choose file. Format as needed. Select the Insert tab, then Chart . This will add a blank chart to...

...Find step-by-step instructions on how to use a water bath in the lab....solution or a 70% ethanol solution to wipe down the inside of the water bath. Pro-Tip To properly maintain...maximum fill capacity is. Look for a “fill” line inside the water bath or read the user manual. If you’...disinfectants designed to be used in water baths with instructions on the bottle, for example, number of drops ...may float in the water bath when they are placed inside. Water bath floats can be used to secure tubes ...before use, make sure that there is enough water inside....

...considering their future careers. Eric J. Perkins, PhD In this installment, we sit down with Senior Scientific...Addgene's online resources. Career Videos Get career insight and advice from real Addgenies! Protocols (Link...after you receive your plasmids from Addgene Instructions for Storing and Handling Plasmids Genomic Deletions...Description Jessica Welch, PhD In this first installment of the Addgene Careers series, we sit down with...Scientific Project Leader Eric Perkins to discuss the various positions he has held in his career, his favorite...

...titers were similar to those reported by these institutions. This protocol is for a 96-well plate with 20...run: 1.5 h Data analysis: 30 min Equipment qPCR instrument Heating plate Pipettors 1–10 µL single channel...Pro-Tips Use a "Universal" SYBR master mix which contains a high-quality DNA polymerase and a blend of dTTP...the final volume (mix with >50 uL if your well contains 100 uL) Use a multichannel pipette to load the...tube. Run the following protocol in your qPCR instrument using SYBR detection: 98 °C 3 min / 98 °C 15 ...2 Sample 5 F Perform data analysis using the instrument’s software. Determine the physical titer of samples...

...specific instructions so be sure to check in with someone familiar with your lab’s instrument before use... often spin at similar speeds to their smaller cousins, but can hold much larger containers. Ultracentrifuges...you may need to fill the blank based on weight instead of volume. Pro-Tip For microfuge tubes, place them... hum. If you hear loud noises or the centrifuge begins to wobble or shake, this could indicate an unbalanced...

...modified for other targets. Primers and a probe against the cellular ribonuclease P/MRP 30 kDa subunit ...ensure that you are in compliance with your institution’s biosafety regulations. Last Update: July 7, ...well untransduced (add 150 µL of DMEM complete instead of a viral dilution). Remember, this is an additional... to the GeneJet Genomic DNA Purification Kit instructions. Determine the concentration of each sample ... To ensure that the droplets are not disrupted, insert the pipette tips directly in the center of the ... To ensure that the droplets are not disrupted, insert the pipette tips and gently touch the bottom of...

... Gibson and colleagues at the J. Craig Venter Institute developed a novel method for the easy assembly...strong secondary structures in the homology region. Hairpins in this region can significantly reduce the efficiency...annealed DNA fragments. Taq DNA Ligase - covalently joins the annealed complementary DNA fragments, removing...for 1 hour at 50 °C or follow manufacturer's instructions. You can purchase master mix or make your own...into the assembly and is used when the part to be inserted is too long to include on overlapping PCR primers...

...protocol describes how to run an indirect ELISA against a purified antigen to demonstrate the expected ...indirect Enzyme-Linked ImmunoSorbent Assay (ELISA) against a purified antigen to validate an antibody. This... the antigen and how to probe with an antibody against the target to show a dose response. Sharing speeds...several days by incubating certain steps at 4 °C instead of room temperature or 37 °C. The protocol notes...minutes. Absorbance was read at 450 nm and plotted against the micrograms of antigen loaded. Last reviewed...

...capabilities. Note: See manufacturer’s instructions for specific instructions about extension time and temperatures...reference page on how to design primers for detailed instructions and check out our protocol on plasmid cloning...each ingredient specifically do? Template DNA: Contains the portion of DNA that we are interested in amplifying...

...depending on the insert. Please ensure that you are in compliance with your institution’s biosafety regulations... To ensure that the droplets are not disrupted, insert the pipette tips directly in the center of the ...Pro-Tip To ensure that the droplets are not disrupted insert the pipette tips and gently touch the bottom of...

...LiCl selectively precipitates RNA versus DNA or proteins. Add the correct amount of 7.5 M LiCl solution...downstream applications. To improve yield of RNA, instead of incubating at -20°C for 1 hour, you can try ...

...For each T-175 flask: Aspirate culture media and rinse once with 10 mL of PBS. Aspirate PBS and add 2 mL... at ~80% confluence. Aspirate culture media and rinse once with 60 mL PBS. Aspirate PBS and add 35 mL ... then transfer the cells into a sterile bottle. Rinse the CS2 with 100 mL of DMEM complete medium and ...Transfer cells and media into 500 mL conical vessels. Rinse CS5 once with 100 mL of PBS and add to the harvested...

...annealed oligos with cut vector in molar ratios (vector:insert) between 4:3 and 1:6 in a standard ligation ...ligation reaction (ex. to ligate an annealed oligo insert of 50bp in length into a 5kb vector, mix 100ng of the... multiple colonies for mini-prepping and verify insert by sequencing. Reference Page Top Index...

...pre-mixed LB agar powder, use the suggested amount, instead of the other dry ingredients above. Media without...in a shaking incubator. Note: Some plasmids or strains require growth at 30°C. If so, you will likely ... to be in the log phase of growth. Check the instructions for your specific protocol and conduct an OD600...

...Creating Bacterial Glycerol Stocks Store bacterial strains or plasmids for long term use Watch the Video! ...Video! Western Blot Separate and detect specific proteins Watch the Video! Immunocytochemistry Use antibodies...the Indirect ELISA Method Run an indirect ELISA against a purified antigen to demonstrate the expected ...

...of light hitting the sample or b) using dyes or stains that add color to some components of the sample..., be sure to record the magnification used, any stains used in your sample, and sample information so ...future. Conclusion Like any important laboratory instrument, you should be sure to take care of your microscope...