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...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...

...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...

...gel extraction of either the original sample or the amplified sample, followed by reamplification of the...tens to millions of different plasmids in a single sample. They are often used for screening, barcoding, ...appear to contain sufficient DNA for use, pool samples, and continue with analysis. “Sufficient” DNA will...

...before you place the sample at -80°C. Frozen tubes are hard to write on and samples stored for long periods...glycerol. You can prepare the glycerol stock the same time you prepare your plasmid DNA. In the morning...

...Pipetting Technique Learn how to pipette multiple samples at once using a multichannel pipette and avoid ...bands out of the agarose gel and purify the DNA samples. This is a commonly used technique for molecular... Your guide to the MTA process when requesting samples from Addgene Technology Transfer How to Order A...

...optimal fixation method will vary depending on the sample type and the protein of interest. You may need ...expected and specific, include a positive control sample that you know expresses the protein, such as cells...the protein of interest, and a negative control sample such as cells that do not express the protein of...

...of DNA Samples Add an equal volume of TE-saturated phenol-chloroform to the aqueous DNA sample. Pro-Tip...remaining contaminant proteins and RNase A from the DNA sample. When phenol is mixed with the aqueous solution...

...absorbed. The spreading of cells can be done in the same way as the antibiotic, using either a bent micropipette...over-agar for selection of transformed E. coli . Sample Data Selection of E.coli on LB-agar using different...

...two different shRNAs that target the same gene can produce the same phenotype will alleviate concerns about...boiling water. If using a PCR machine, incubate the sample at 70°C for 10 minutes then slowly cool to room...

...double digest (digesting with two enzymes at the same time), you will need to determine the best buffer...are digesting a large number of plasmids with the same enzyme(s) (for instance, in a diagnostic digest)...

...protocol outlines the seeding of the cells at the same time as the virus-mediated transduction. For some... titer, take the average of multiple dilutions. Sample Data Figure 1: A549 cells were transduced with ...