...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. Note: Loading...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...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...band), you can infer the size of the DNA in your sample lanes. For more details on doing diagnostic digests...

...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...

...plasmids. Plasmids within a given library have the same backbone, but they express or target different genes...Perform Next Generation Sequencing (NGS) on a control sample (no selection) In parallel, apply library Apply...

...While both lentiviruses and γ-retroviruses use the same gene products for packaging (i.e., Gag, Pol, and.... Return to Top Glossary Plasmid Type Element On Same Plasmid as Transgene? Purpose Envelope VSVG in trans...

...toolkit for human gene therapy. Wu Z, Asokan A, Samulski RJ. Mol. Ther. 2006 Sep;14(3):316-27. Epub 2006...independently of DNA synthesis. McCarty DM, Monahan PE, Samulski RJ. Gene Ther. 2001 Aug;8(16):1248-54. PubMed ...

...CCCTTGAACCTCCTCGTTCGACC (MSCV) Murine stem cell virus, same as MSCV, forward primer pMRB101-F AAGATGCAGGCAGCTGAGTT...GATTTAATCTGTATCAGG (Invitrogen) 3' of MCS in pTrcHis vector, same as pBAD-R, reverse primer Puro-F GCAACCTCCCCTTCTACGAGC...

...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...

...side of it into any plasmid that has the same sites in the same orientation within its multiple cloning...

...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...

...make it possible to combine multiple opsins in the same experiment. For example, you could use different...different colors of light to activate/silence the same neuronal population, or activate different neural populations...

...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...

...performed at Addgene on the A549 cells described in our sample data , each 96-well plate gave rise to 15–20 monoclonal...solution will be used to seed the 96-well plate. Sample Calculation First, use a hemocytometer to quantitate...plate for individual cells, which should be on the same focal plane. Scan the entire plate and make note...highest or lowest transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal cell lines...