...of cut insert and vector to use for the ligation reaction. The volume of vector DNA and insert DNA used...standard cloning (where the insert is smaller than the vector) a 3 insert : 1 vector molar ratio will ...vector and insert DNA to use. Combine the following in a PCR or Eppendorf tube: Vector DNA Insert DNA Ligase...transformation reagents Optimizing the Vector:Insert Ratio Although a 3:1 insert to vector ratio is usually sufficient...construction of a recombinant plasmid is connecting the insert DNA (gene or fragment of interest) into a compatibly...join the nucleotides together. After ligation, the insert DNA is physically attached to the backbone and ...called "sticky ends", are what allow the vector and insert to bind to each other. When the sticky ends are...

...choose enzymes that: Flank your insert, but do not cut within your insert Are in the desired location in...commonly used. Follow the manufacturer’s instructions. Isolate your insert and vector by gel purification Run...DNA . Ligate your insert into your vector Conduct a DNA Ligation to fuse your insert to your recipient...plasmid to insert. It is also important to set up negative controls in parallel. For instance, a ligation...cut elsewhere on the plasmid Will result in your insert being in the correct orientation in the recipient...of your gene!) Are in frame with tags or fusion proteins in the recipient plasmid (if you are creating ...specifically designed test digest later to verify that the insert was cloned in the correct orientation. If you cannot...

...the gel can indicate if the plasmid contains the expected size insert. By selecting the appropriate enzyme...manufacturer's instructions) Gel loading dye Electrophoresis buffer Verifying Total Plasmid Size -OR- Insert and ...plasmid. Verifying Insert Orientation by Restriction Digest If you clone an insert into a vector by a ...orientation of the insert. By choosing an enzyme (or enzymes) that cuts within the insert, but significantly...diagnostic digest to verify plasmid size, verify insert orientation, and more. Protocols...restrictions sites. Often, the size of the plasmid insert and vector backbone are known and thus this technique...the entire construct or excise some or all of an insert from it. Before beginning your diagnostic digest...

...commonly used. Follow the manufacturer’s instructions. Isolate Your Insert and Vector by Gel Purification Run...DNA . Ligate Your Insert Into Your Vector Conduct a DNA Ligation to fuse your insert to your recipient...plasmid to insert. It is also important to set up negative controls in parallel. For instance, a ligation... to choose enzymes that: Do not cut within your insert. Are in the desired location in your recipient ...our cDNA into the recipient plasmid. Remember to insert your DNA in the correct orientation in the recipient...Ppurify the PCR Product Run PCR to amplify your insert DNA. It is important to use a high fidelity taq...of DNA). Once you have cut out and purified your insert and vector bands away from the gel, it is important...

...white precipitate will be formed which contains the bacterial proteins and genomic DNA. Incubate tube on ice...tube for 5 min at 12,000 g. Notes: Pellet contains proteins, cell fragments, salt and other extra particles...min at 4°C. Notes: Pellet contains the precipitated DNA. Supernatant contains residues, salts, and water...If you will be using a kit, follow the kit's instructions. If you want to perform plasmid purification... This step brings the pH back down causing the proteins and genomic DNA to precipitate, while leaving ... smaller plasmids free in solution. Pellet the proteins and genomic DNA by centrifugation, and remove ...that has been purified away from the bacterial proteins and genomic DNA. Depending on the method used,...

...your institution’s Bio-Safety office to receive permission and institution-specific instructions. You ...bacteria D. Screening for Inserts D.1 Recommended materials D.2 Screening for inserts E. Producing Lentiviral...Consortium has built a library of shRNAs directed against 15,000 human and 15,000 mouse genes. Addgene is...selection. Figure 1 : Map of pLKO.1 containing an shRNA insert. The original pLKO.1-TRC cloning vector has a 1.9kb... Negative control vector containing non-hairpin insert. pLKO.1 – scramble shRNA Negative control vector...targets for your gene. For example, the Whitehead Institute for Biomedical Research hosts an siRNA Selection...Additional design rules can be found at the Broad Institute's TRC shRNA Design Process . To minimize degradation...

...Appropriate restriction enzyme (see manufacturer's instructions for proper ammount) Approrpriate restriction...restriction digest buffer (see manufacturer's instructions) Gel loading dye Electrophoresis buffer Pipet tips Procedure...an appropriate reaction buffer by reading the instructions for your enzyme. Pro-Tip If you are conducting...for 1 hour. Always follow the manufacturer’s instructions. Pro-Tip Depending on the application and the... the reaction, incubation time can range from 45 mins to overnight. For diagnostic digests, 1-2 hours ... involve incubating the reaction at 70 °C for 15 mins, or purifying the DNA via a purification kit, such... cleanup kit . See the enzyme manufacturer's instructions for more details. To visualize the results of...

...competent cells in your hand instead of on ice Reduce step 4 from 20 - 30 mins to 2 mins on ice before heat-shock...many genetic modifications to create bacterial strains that can be more easily transformed and that will...efficiency, we recommend that you follow the instructions that came with your competent cells. Pro-Tips...out of -80°C and thaw on ice (approximately 20-30 mins). Remove agar plates (containing the appropriate...be approximately 10-fold lower for ligation of inserts to vectors than for an intact control plasmid. ... the competent cell/DNA mixture on ice for 20-30 mins. Heat shock each transformation tube by placing ...bacteria time to generate the antibiotic resistance proteins encoded in the plasmid backbone so that they will...

...weight proteins and higher percentage acrylamide when immunoblotting low molecular weight proteins. This...Transfer for 5–6 min for proteins <30 kDa. Transfer for 8–10 min for proteins >150 kDa. Select Start Run...Introduction Western blot is a technique used to separate proteins by size followed by detection using antibodies...Incubate with secondary antibody Video Watch this instructional video to learn how to use western blotting to... Before Starting Refer to the manufacturer's instructions for additional information specific to your ... of interest. RIPA buffer is suitable for most proteins but more stringent buffers and a sonication step...step may be required for hard to extract proteins such as those in the nucleus. Incubate on ice for 30...

...poured gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely ... a rocker for 20-30 mins, replace EtBr solution with water and destain for 5 mins. Using any device that...flask. See TAE Recipe . Pro-Tip TBE can be used instead of TAE, labs usually use one or the other, but ...comfortably keep your hand on the flask), about 5 mins. Optional : Add ethidium bromide (EtBr) to a final... need to soak the gel in EtBr solution and then rinse it in water before you can image the gel. Pour the...you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the...causing it settle to the bottom of the gel well, instead of diffusing in the buffer. Once solidified, place...

...: 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...