...but you should ensure that the bases do not result in the formation of a hairpin structure within your primer...You should select an annealing temperature based on the melting temperature (Tm) of the portion of the primer...TGCTTAGCGGCCGCTCAGTACTTCGAGATATGCCA-3’. Experimental Procedure Run PCR and Ppurify the PCR Product Run PCR to...you can use a pretty wide range of annealing temperatures, but you may need to increase your primer length... phosphatase) are commonly used. Follow the manufacturer’s instructions. Isolate Your Insert and Vector...Proceed with the transformation according to the manufacturer’s instructions for your competent cells. For ...colonies, you should conduct a positive control to ensure that your transformation worked. You could also...

...with their competent cells) to ensure that your transformation procedure is working. TIP: Sometimes less... competent cells. This is a relatively simple procedure and is useful for performing low efficiency transformations...media Competent cells DNA you'd like to transform Procedure Take competent cells out of -80°C and thaw on ... from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator...control plasmid. Incubate the competent cell/DNA mixture on ice for 20-30 mins. Heat shock each transformation...allowing you to recover all transformants. If the culture volume is too big, gently collect the cells by ...electro-magnetic field is applied to the cell/DNA mixture to induce membrane permeability. To do this you...

...DNA fragments at once. Procedure Design your plasmid and order primers (see figure to the right). When designing...sequences on the ends (sequences A and B in the figures). These identical sequences can be created via ...to the target sequence. Avoid strong secondary structures in the homology region. Hairpins in this region...three different enzymes within a single buffer mixture and an optional SSB protein to improve accuracy...Exonuclease and furthermore reduces secondary structure of ssDNA. (Rabe & Cepko, 2020). Incubate the mix... mix for 1 hour at 50 °C or follow manufacturer's instructions. You can purchase master mix or make your... DNA molecules up to several hundred kilobases. Nature Methods , 6(5), 343–345. https://doi.org/10.1038...

...buffer, it will save you time in future steps. Experimental Procedure Digest your DNA Set up restriction...subcloning), including design and experimental procedures. Protocols...orientation on your target vector. If you are not sure what vector to use, you can check out our Empty ... for short). You might need to express YGOI in cultured mammalian cells. The problem is that the only ... phosphatase) are commonly used. Follow the manufacturer’s instructions. Isolate your insert and vector...your bacterial strain of choice. Follow the manufacturer’s instructions for your competent cells. For ...colonies, you should conduct a positive control to ensure that your transformation worked. You should also...

...Foil Before Starting Warm reagents to room temperature. Procedure Section 1: Prepare the Antigen Standard...incubating certain steps at 4 °C instead of room temperature or 37 °C. The protocol notes when there are different...microplate shaker set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section 3: Primary antibody...microplate shaker set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section 4: Secondary antibody...microplate shaker set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section 5: TMB reaction...seal and wrap in foil. Incubate plate at room temperature on a microplate shaker set at 400 rpm for 15–...Solution directly to the wells to stop the reaction. Measure the absorbance of each well at 450 nm on the spectrophotometer...

...the basic steps for fixing and staining cells in culture with a primary antibody against a target protein...the basic steps for fixing and labeling cells in culture with a primary antibody against a target protein... conical tubes Before Starting Refer to the manufacturer's instructions for additional information specific...stock solution into 5 mL PBS. Protect from light. Procedure Section 1: Seeding cells Place a sterile poly-...coated coverslip in each well of a 24-well cell culture treated plate. Seed 5 x 10 3 HeLa cells per well...platform. Permeabilize cells for 10 min at room temperature ( RT ) on a rocking platform in 500 µL permeabilization...concentration will vary between antibodies. Review the manufacturer's instructions before starting your experiment...

...between them. Sample Data Figure 1: Example of a valid 8-point standard curve. Figure 2: Example of the amplification...additional 10 samples (n+10 – the additional amount will ensure that there is enough master mix for all samples...0.15 μL 15 μL Nuclease Free Water 4.7 μL 470 μL Procedure Prepare a plasmid stock of 2 x 10 9 molecules/... of the sample dilution series is critical. Make sure to pipet each dilution up and down at least 10 times...standard curve and the sample dilutions. Pro-Tip Make sure that the qPCR is valid by checking to the following... be seen. The presence of a second peak at a temperature of ~70–75 °C usually indicates the presence of...

...solution to mix for 10 min and then recheck the pH to ensure that it has not drifted. Filter the solution through.... Thaw a new vial of cells after 30 passages. Procedure Trypsinize and resuspend the HEK293T cells from...80% confluence. For each T-175 flask: Aspirate culture media and rinse once with 10 mL of PBS. Aspirate.... Cells should be at ~80% confluence. Aspirate culture media and rinse once with 60 mL PBS. Aspirate PBS...the OptiMEM + DNA + PEI solution to the CS5. Make sure that all five layers are covered with media. 293T...should always be added away from the cells (not poured on them) and can be adjusted by carefully tilting...proceeding with the purification protocol. Sample Data Figure 1: HEK293T cells at various confluencies. This ...

...Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions). ...end and a different enzyme on the 3' end). This ensures that the insert will be added in the correct orientation...reaction, scale the reaction size as necessary - being sure to increase the amount of buffer proportionally....C. Whenever you need to set up ligations in the future you can thaw a new tube that you know has only ...using "high concentration" ligase, 5min at room temperature is enough. For trickier ligations (such as ligation...

... the UV exposure of the DNA. Therefore, it is a bad idea to use a gel imager to take a picture of the ...isolate and purify DNA fragments based on size. The procedure starts with standard agarose gel electrophoresis...have run your gel, move it to an open UV box (be sure to wear proper UV protection - especially for your...QIAquick Gel Extraction Kit . Always follow the manufacturer's instructions. Note: It is usually important...

...Microcentrifuge tubes, Neptune 3745.X 10 cm tissue culture dish, Corning 430167 Hydrochloric acid Sodium hydroxide...solution to mix for 10 min and then recheck the pH to ensure that it has not drifted. Filter the solution through...the best ratio of mass DNA:mass PEI determined. Procedure Seed HEK293T cells at 3.8x10 6 cells per plate...plate in DMEM complete in 10 cm tissue culture plates. Incubate the cells at 37 °C, 5% CO 2 for ~20 h. Gently...gently flicking the diluted DNA tube. Incubate the mixture 15–20 min at RT. Carefully transfer the transfection...

...Deionized water Before Starting Refer to the manufacturer's instructions for additional information specific...antibody for primary antibodies raised in a mouse. Procedure Section 1: Lyse cells Centrifuge 5 x 10 6 cells...of the device. Select the desired method and make sure the parameters are correct. Transfer for 5–6 min... will vary between antibodies. Refer to the manufacturer's information before running your experiment....your antibody to determine the optimal dose. To ensure that your antibody is both functioning as expected...

...amplified should be within 1-10 kB in size. The structure of the primer should be relatively simple and ...and contain no internal secondary structure to avoid internal folding. One also needs to avoid primer-primer...disrupts the amplification process. When designing, if unsure about what nucleotide to put at a certain position...content Start and end with 1-2 G/C pairs Melting temperature (Tm) of 50-60°C Primer pairs should have a Tm...

...add antibiotic to a plate after it's already been poured and solidified....Transformation Recovering Plasmid DNA from Bacterial Culture Introduction This protocol describes methodology...resistance gene (or other antibiotic resistance) Procedure Day 1 Prepare carbenicillin to a concentration...your petri plate. Incubate the plate at room temperature for at least 30 minutes with the lid on to give...

...to add short tags or shRNAs to any vector (the procedure will simply differ in terms of primer design)....be performed enzymatically later. Experimental Procedure Digest and purify vector While waiting for your...bench top) allowing for slow cooling to room temperature (~45 minutes). Method #2 Place mixed oligos in...into your favorite competent bacteria and plate. Be sure to pick multiple colonies for mini-prepping and ...

...Introduction Sequence verification of important plasmid features (such as the gene/insert, fusion proteins, point...sequencing primers, including: Is the primer unique? Make sure the primer only anneals once within the entire construct...mis-called? Open the trace file and use the search feature in the program to locate the incorrect sequence...sequence. Look at the peaks in the area and make sure they are justifiable peaks. For instance, in the trace...

...generally considered biosafety level 2+. Please ensure that you are in compliance with your institution...Microcentrifuge tubes, Neptune 3745.X 6-well tissue culture treated dish, Corning 3516 15 mL conical tubes,... supernatants to multiple freeze-thaw cycles. Procedure Seed 75,000 cells into each well of a 6-well dish... the average of multiple dilutions. Sample Data Figure 1: 293T cells were transduced with a range of dilutions...

...Microcentrifuge tubes, Neptune 3745.X 6-well tissue culture treated dish, Corning 3516 15 mL conical tubes,...transduction, and not during the cell seeding step. Procedure Before beginning a colony formation assay, the... the average of multiple dilutions. Sample Data Figure 1: A549 cells were transduced with the indicated...then stained with 0.1% crystal violet and counted. Figure 2: A549 cells were transduced with the indicated...

...so tight that you cannot perform your duties. Be sure to try out different sizes to know your fit. It ...protocol goes a long way. It is the easiest way to ensure that you’re doing your work in the safest way possible...note that you do so at your own risk; you should ensure that any local guidance is also adhered to. None...

...amounts) Appropriate restriction enzyme (see manufacturer's instructions for proper amount) Approrpriate...Approrpriate restriction digest buffer (see manufacturer's instructions) Gel loading dye Electrophoresis buffer... dig one out of the freezer and you are not 100% sure it is what you are looking for, but you have a map...