...layout is complete, select 'Run' to begin the droplet reading. When the droplet reading is complete, export...concentration of each sample on a spectrophotometer. Prepare 25 ng/µL stocks of each sample. Samples can be used...the droplet generator. Close the lid and wait for the droplets to be generated. Once the droplets have...the droplets. Transfer the droplets to a prechilled PCR plate. Pro-Tip To ensure that the droplets are...constant across samples. In the RRE example below, 2-fold serial dilutions of a sample were loaded in ...Lentivirus sample data, RPP30 positive (green) and negative (black) droplets Sample ID Plasmid ID Dilution...incubating with 200 µL TrypLE for 1–2 min. Resuspend cells in 500 µL DMEM complete and transfer to a microcentrifuge...

...layout is complete, select 'Run' to begin the droplet reading. When the droplet reading is complete, export...the droplet generator. Close the lid and wait for the droplets to be generated. Once the droplets have...the droplets. Transfer the droplets to a prechilled PCR plate. Pro-Tip To ensure that the droplets are... ddPCR sample data Sample Dilution Concentration Titer (GC/mL) Average Titer (GC/mL) AAV Sample 1 6400000... used for master mix preparation and droplet generation (droplet generation BSC) to ensure no contamination...tube as shown below. For 8 samples prepare enough master mix for 9 samples. ITR Master Mix Volume 9X Volume... the PCR tubes. Generate the Droplets Bring the PCR tubes to the droplet generation BSC. Without disturbing...

...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...between them. Sample Data Figure 1: Example of a valid 8-point standard curve. Figure 2: Example of the amplification...standards and samples in duplicate at least If possible, include an AAV reference sample of known titer...error and variability Mix samples very well by pipetting back and forth multiple times at each step Reagent...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...

... 10 seconds. Incubate sample(s) for 15 minutes on ice and centrifuge the sample(s) for 15 minutes at 12,000...RNA. Store your RNA sample(s) at -80 °C to prevent RNA degradation and avoid multiple freeze-thaw cycles...cycles. Pro-Tip To avoid multiple freeze-thaw cycles of your entire RNA sample, consider making smaller aliquots...RNA.Store your RNA sample(s) at -80 °C to prevent RNA degradation and avoid multiple freeze-thaw cycles...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 ...

...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...area of all bands in the lane Example for AR0018 (lane 2 in Figure 1): Sample Peak 1 (contaminant) Peak 2...

...binding capacity. If your sample exceeds the capacity, divide the sample among multiple columns. For information...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...similar outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted...

...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...

... which you need to spin your samples. For example, spinning down samples in 1.5 mL microfuge tubes at ...liquid sample. It achieves this by using centrifugal force, which is generated by spinning the sample at ... with water to the same volume as your samples. If your samples are significantly higher density than ...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 ...speed needed for your experiment. Procedure An example of a balanced tabletop centrifuge, with tubes evenly...

...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...similar outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted...blotting to visualize a protein from cells or tissue samples. Equipment Microcentrifuge 0.5–10 µL single channel...

...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...your sample lanes. For more details on doing diagnostic digests and how to interpret them please see the...). Microwave for 1-3 min until the agarose is completely dissolved (but do not overboil the solution, ...the final percentage of agarose in the gel. Many people prefer to microwave in pulses, swirling the flask...room temperature for 20-30 mins, until it has completely solidified. Pro-Tip If you are in a hurry, the...

...trypsin by adding 10 mL of DMEM Complete. Pipet back and forth vigorously multiple times to obtain a single ...Thermo Fisher 31985-070 0.05% Trypsin/EDTA (e.g. TrypLE, Thermo Fisher, 12605010) 1X PBS pH 7.4 without... mM NaCl, 2 mM MgCl2) Reagent Preparation DMEM Complete : 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...Chloride Close the bottle and mix by inverting multiple times Adjust pH to ~8.5 Filter sterilize through...-175 flasks. Adjust volume to 300 mL with DMEM complete media and mix. Seed all cells in 1 CS2. Return...CS2 to help detach the cells, add 200 mL of DMEM complete media, then transfer the cells into a sterile ...sterile bottle. Rinse the CS2 with 100 mL of DMEM complete medium and pool with the cells harvested in the previous...

...more accurate titer, take the average of multiple dilutions. Sample Data Figure 1: A549 cells were transduced...Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...recommended that lentiviral supernatants be subjected to multiple freeze-thaw cycles. This protocol outlines the...colony formation assay. Prepare a batch of DMEM complete containing 10 μg/mL polybrene by diluting 20 μL...fold serial dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene: Dilution Volume...Lentivirus or Previous Dilution (μL) Volume of DMEM Complete Containing 10 μg/mL Polybrene (μL) Volume of Virus...follows: Dilute 7,000 cells into 9.45 mL of DMEM complete containing 10 μg/mL polybrene. Mix well by pipetting...

...more accurate titer, take the average of multiple dilutions. Sample Data Figure 1: 293T cells were transduced...Lentivirus is generally considered biosafety level 2+. Please ensure that you are in compliance with your institution...Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...recommended to subject lentiviral supernatants to multiple freeze-thaw cycles. Procedure Seed 75,000 cells...follows: Dilute 525,000 cells into 14 mL of DMEM complete. Mix well by pipetting or inverting. Aliquot 2... Prepare dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene. Note, this protocol...Volume of Lentivirus Stock (μL) Volume of DMEM complete (μL) Volume of 10mg/mL polybrene (μL) 1:10 150...

... original sample or the amplified sample, followed by reamplification of the DNA, but please note that...single sample. They are often used for screening, barcoding, or other high throughput multiplexed experiments...process my Addgene pooled library? Introduction Please refer to our pooled library material pages for ...targeting different genes in an organism's genome, for example. Amplification is usually necessary to produce ...basic knowledge of the library being amplified. Please see our protocol on Bacterial Transformation . ...electrocompetent cells on ice for 15-20 minutes or until completely thawed. Chill a box of 200 µL micropipette tips...Electroporation involves the use of high voltages, please use caution when activating pulse and follow all...

...between samples. In addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each...choice with minimal limitations. Background In its simplest form, PCR based cloning is about making a copy...easily cloned into a plasmid of interest. For this example, we will describe how to copy a cDNA from one vector...location in your recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere ...same buffer, as this will save time later. In our example, we will use EcoRI and NotI to ligate our cDNA ...start codon (ATG) to the stop codon (TGA, in this example). Assuming you are amplifying from plasmid DNA...design is similar, but we need to use the reverse complement to get PCR amplification. We can start similarly...

... to follow, please contact your institution’s safety office. For more information, please see Addgene'...cloning vector available to the scientific community. Please cite Moffat et al., Cell 2006 Mar; 124(6):1283-...sequence), while the EcoRI site is preserved. For a complete map of pLKO.1 containing the 1.9kb stuffer, visit... nuclear import of the vector’s preintegration complex in the transduced cells. hPGK Human phosphoglycerate... set of top-scoring targets for your gene. For example, the Whitehead Institute for Biomedical Research...genes. TIP: Addgene recommends that you select multiple target sequences for each gene. Some sequences...AATTCAAAAA—21bp sense—CTCGAG—21bp antisense 3’ For example, if the target sequence is (AA)TGCCTACGTTAAGCTATAC...

...DNA molecule (single gene perhaps) to be copied multiple times by Taq Polymerase. From a single copy of...can create thousands of identical copies using a simple set of reagents and a basic heating and cooling...denaturation, annealing, and synthesis. After the PCR is complete, the product can be verified based on size by ...Sterile dH 2 O (variable) Note: If you are doing multiple PCRs, save time by creating a “master mix,” which... extra to account for pipetting error. In this example, we have 7 different reactions (7 unique primer...placing it in the PCR machine. Diluting Primers Most people order primers from a company, which synthesizes...to the number of nanomoles of DNA times 10. For example, if your lyophilized DNA is 38.5nm, add 385µl of...

...the lentivirus in DMEM complete + 10 µg/mL polybrene. Note, this is just a sample of possible dilutions...puromycin, blasticidin) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...recommended that lentiviral supernatants be subjected to multiple freeze-thaw cycles. Procedure Before beginning...kills all of the cells. Prepare a batch of DMEM complete + 10 µg/mL polybrene by diluting 20 µL of 10 mg...Dilution Volume of Lentivirus (μL) Volume of DMEM complete + 10 µg/mL polybrene (µL) 0 0 500 1:5 300 200 ...,000 cells into a total volume of 7 mL of DMEM complete + 10 µg/mL polybrene. Mix well by pipetting or...all the media in these wells was made with DMEM complete + 10 µg/mL polybrene, the final concentration ...

...advisable to set up multiple reactions by holding the vector concentration fixed across multiple concentrations...from their recognition sequence. This can create multiple distinct overhangs with a single enzyme, and remove...counter-selection for negative clones as in the example shown here. The manufacturer of an LIC-specific...respective primers. Below we use pNIC28-Bsa4 as an example of LIC experimental design. Search Addgene's collection... Your Primers Primer design for LIC is often as simple as using the backbone manufacturer's suggested ...different leader sequences, but operate on the same principle (homologous to the first G on 3'-5' strand from... PCR primers. Step 2: Linearize Vector In this example, the vector is cut at two points by the enzyme ...

...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...