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...empty NTC C AAV reference Sample 3 D E Sample 1 Sample 4 F E Sample 2 Sample 5 F Perform data analysis...most samples. If sample is expected to have a titer <1 x 10 12 GC/mL, use dilutions 3–6 If sample is expected...standards and samples in duplicate at least If possible, include an AAV reference sample of known titer...Count the number of samples (n) and prepare master mix for an additional 10 samples (n+10 – the additional...that there is enough master mix for all samples). Each sample requires 15 μL of master mix. Pro-Tips Use...pipetting error and sample evaporation. Make the master mix after all the samples have been added to the...virion). 5 μL sample + 39 μL H 2 O + 5 μL 10X DNase buffer + 1 μL DNase Gently mix sample (do not vortex...

...tube filled with water to the same volume as your samples. If your samples are significantly higher density...liquid sample. It achieves this by using centrifugal force, which is generated by spinning the sample at ... which you need to spin your samples. For example, spinning down samples in 1.5 mL microfuge tubes at ...the pellets form in approximately the same place in each sample. Place the centrifuge’s internal lid on...centrifuge to separate different components in a liquid sample. Protocols... containers, spin at different speeds, or keep samples at specific temperatures. This protocol will cover...for the biosafety level of the lab space Reagents Sample to be centrifuged Water Background Information ...

...helps concentrate the light onto the sample, and then through the sample. However, some of that light won’...hitting the sample or b) using dyes or stains that add color to some components of the sample but not others... power objective to find your sample. Place your slide (or other sample type) on the microscope stage....so that the light shines onto your sample. To ensure that your sample is adequately illuminated, look through... coarse focus knob to bring your sample into focus. Once your sample is in focus, use the stage arm to... you can easily see your sample. Then use the focus knobs to bring the sample into focus. Pro-Tip As you...magnification used, any stains used in your sample, and sample information so that you or others can interpret...

...of 4X sample buffer to each sample. Add 2 µL 10X reducing agent to each sample. Spin the sample briefly...concentration of your samples based on your sample’s HC + LC area. Example calculation: If the sample HC + LC Area...Invitrogen NP0001 4X NuPage sample buffer, Invitrogen NP0007 10X NuPage sample reducing agent (500 mM dithiothreitol...microcentrifuge. Heat the samples for 10 min at 100 °C in a heat block. Spin the sample briefly in the microcentrifuge...one lane before loading samples. Load 20 µL of each recombinant antibody sample to the appropriate well... mobility of the bands (sample AR0016 in Figure 1) indicates that the samples may not have been processed... SimplyBlue SafeStain) and ImageJ software. The sample is separated by denaturing polyacrylamide gel electrophoresis...

...finite binding capacity. If your sample exceeds the capacity, divide the sample among multiple columns. For...4 mL of recombinant antibody sample. Pro-Tip If the volume of the sample is greater than 4 mL , then divide... Slowly apply the sample to the center of the compact resin bed. Pro-Tip For sample volumes <1.5 mL top...the sample with 0.2 mL PBS to aid protein recovery. Allow the resin bed to fully absorb the sample. Centrifuge...Spectrophotometer to see if the sample has reached the desired concentration. If the sample concentration is still... to see if the sample has reached the desired concentration. Gently transfer the sample from the reservoir...Determine the protein concentration of the pooled sample on the NanoDrop Spectrophotometer using the A280...

... 10 seconds. Incubate sample(s) for 15 minutes on ice and centrifuge the sample(s) for 15 minutes at 12,000...Always wear gloves, and whenever possible, keep RNA sample and reagents cold and work quickly to reduce RNA...use 1 mL of Solution D per 1 X 10 7 cells. Allow sample(s) to sit at room temperature for 5 minutes to ... tissues such as bone, or bacteria/yeast/plant samples will require additional steps to effectively lyse...open the cells. Extract RNA from the homogenized sample(s).Transfer tissue/cell lysate to a 4 mL tube. ...of the tissue/cell lysate. Pro-Tip Having your samples spin at 4°C helps reduce RNA degradation. If you.... You may have to collect twice or more from the same tube, but unlike using a p1000 tip it will give ...

...concentration of each sample on a spectrophotometer. Prepare 25 ng/µL stocks of each sample. Samples can be used...tube as shown below. For 8 samples prepare enough master mix for 9 samples. Component Volume 9X Volume...constant across samples. In the RRE example below, 2-fold serial dilutions of a sample were loaded in ...ddPCR Lentivirus sample data, RPP30 positive (green) and negative (black) droplets Sample ID Plasmid ID ... higher dilutions depending on their particular sample. This protocol was modified from the publication...Starting Thaw the master mix, primers/probe mixes and samples on ice before use. Wipe down all pipettes and surfaces...C until ready to use. Preparing for ddPCR Thaw samples, primers/probe mixes, and master mix on ice. Before...

...duplicate samples on the plate. Subtract the average absorbance of the blank from all of the samples. Plot...concentration of the sample from the standard curve. Determine the volume of sample required to load an...protein for each sample. Pro-Tip The ideal total protein loaded will vary between samples and target proteins...abundance in the sample you will need to load a greater amount of total protein. Prepare sample for loading...Dilute 4X protein loading buffer in the sample to 1X . Boil the samples for 10 min at 100 °C . Section 3: SDS-PAGE...blotting to visualize a protein from cells or tissue samples. Equipment Microcentrifuge 0.5–10 µL single channel...duplicate, dilute 10 µL of standard, blank, and lysate samples into 200 µL of BCA reagent in a 96-well microtiter...

... ddPCR sample data Sample Dilution Concentration Titer (GC/mL) Average Titer (GC/mL) AAV Sample 1 6400000...tube as shown below. For 8 samples prepare enough master mix for 9 samples. ITR Master Mix Volume 9X Volume...prepared for each AAV sample, and the three final dilutions are assayed. The samples that are assayed are... higher dilutions depending on their particular sample. For additional tips on AAV titering using ddPCR...channel 1–10 µL pipette to add 5 µL of each viral sample to Dilution 1 in the 48-well dilution plate and...not push the cap in all the way, just ensure the samples are covered. Bring the PCR tubes to the BSC used...software to set up a new plate layout. Designate the sample name, experiment type, supermix type (ddPCR Supermix...

...steadily, push the sample out and watch as the sample fills the well. After all of the sample is unloaded, .... Loading Samples and Running an Agarose Gel: Add loading buffer to each of your DNA samples and mix well...Note: When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or...difference between the two. Note: Make sure to use the same buffer as the one in the gel box (do not mix different... well. Use 5 µl of loading buffer per 25 µl of sample. Note: Loading buffer serves two purposes: 1) it...glycerol that increases the density of your DNA sample causing it settle to the bottom of the gel well...straight out of the buffer. Carefully load your samples into the additional wells of the gel. Run the gel...

...Calculate your DNA sample concentration and purity using Addgene’s DNA Quantification Protocol....generally used as an indicator of the purity of DNA samples. These days, many labs have a NanoDrop, which is...lab. Spectrophotometer Tips Before measuring any samples, be sure to ‘blank’ the spectrophotometer using...your solvent. If using a NanoDrop to measure your samples, place 1-2µL of mini-prepped DNA onto the pedestal... purity ranges from 1.80-2.00). Repeat for each sample. Notes: Keep in mind that despite the accuracy ...accuracy of the NanoDrop, if two consecutive samples have significantly different concentrations, it is possible... idea to re-zero any spectrophotometer between samples if they are expected to vary significantly in concentration...

...between samples. In addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each...by restriction sites that are also present in the same orientation on your target vector. If you are not...a cDNA from one plasmid to another. However, the same technique can be used to move promoters, selectable...useful to see if one set of enzymes will work in the same restriction enzyme buffer (see (Link opens in a .... If you select enzymes that can function in the same buffer, it will save you time in future steps. Experimental...

...growth. See our sample data section below for positive and negative test results. Sample Data In all cases...Transfer the sterile water (in our case 220 mL) to the same bottle and swirl to form a medium/agar colloid. ...will darken during the autoclave process if your sample has spent at least 10 min at 121 ℃. Use lab tape...the autoclave and run on a setting that gets the sample to at least 121 ℃ under 20 psi for at least 30 ... any weird and wonderful organisms. While your samples are sterilizing in the autoclave, you should prepare...

...contaminating your sample. To accomplish this, it is best to skip lanes between samples and between the ...bands out of the agarose gel and purify the DNA samples. This is a commonly used technique for molecular...the ladder and nearest sample. To minimize the risk of DNA damage, it is best to limit the UV exposure of...will help separate larger bands. 10% Rule For each sample you want to load on a gel, make 10% more volume...

...pipette tip is so that the same pipette can be used for measuring different samples without cross contamination...contamination as long as the tip is changed between samples. Tips can come loose in a bag, or can come preloaded...on the Pipette Although each pipette performs the same function, the numbers are read differently on each...correct amounts of liquid. If you are pipetting the same amount of liquid into different tubes or into wells...

...Add your sample. Spin at 3500 rpm for 5–8 min at 4 °C, discard the flow through. Add more sample and spin...After each spin, add more formulation buffer and sample and make sure to pipet back and forth a few times..., or aliquot and store at -80 °C for long term. Sample Data Figure 1: Iodixanol gradient before ultracentrifugation...Grieger, Joshua C., Vivian W. Choi, and R. Jude Samulski. "Production and characterization of adeno-associated...

...) (Link opens in a new window) (3,180 cm 2 - the same surface area as 21 x T-175 flasks). Cell stacks ...0.08 μg/bp Therefore, for each plasmid we need: Sample Calculations RepCap: 0.08 μg/bp × 7,265 bp = 582.1...overnight at 4 °C if needed. Transfer the entire sample to 3 x 500 mL conical bottles and centrifuge at...round of sonication to avoid overheating of the sample. Mix well between rounds of sonication. Sonicate...before proceeding with the purification protocol. Sample Data Figure 1: HEK293T cells at various confluencies...

...be resuspended before sampling. Gently swirl the flask 5–10 times before sampling. Transfer 10 µL of trypan...the PEI-MAX and recombinant antibody plasmid DNA sample to the biosafety cabinet and incubate for 1 h at...significantly and should be empirically determined for your sample. Typical ratios may range from 1:1 to 1:6. Add ...

...between samples. In addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each...about making a copy of a piece of DNA and at the same time adding restriction sites to the ends of that...restriction enzymes that can both function in the same buffer, as this will save time later. In our example...

... unknown sample (don’t forget to factor in that dilution when calculating the original sample concentration...is a suggested starting point. If your unknown sample’s absorbance falls above the range of the standard...concentration!). Sample Data Figure 1: The plate was coated with serial dilutions of human recombinant purified...