...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...

...strand completely; it is essential, however, that the 3’ end of the primer corresponds completely to the...reaction. One needs to design primers that are complementary to the template region of DNA. They are synthesized...sequences on the template molecule (must be complementary to template strand). However, primers do not...primers are mainly used for amplifying a small, simple fragment of DNA. On the other hand, a long primer...primer is used to amplify a eukaryotic genomic DNA sample. However, a primer should not be too long (> 30...The structure of the primer should be relatively simple and contain no internal secondary structure to ... of each other Primer pairs should not have complementary regions Note: If you will be including a restriction...

...contaminating your sample. To accomplish this, it is best to skip lanes between samples and between the ...How do you get better resolution of bands? A couple simple ways to increase the resolution (crispness) ...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...several microliters can be lost in pipetting. For example, if you want to load 1.0 μg in 10μL, make 1.1μg...

...growth. See our sample data section below for positive and negative test results. Sample Data In all cases...bacteria containing the plasmids of interest (for example, fewer satellite colonies will grow). However, ...on the number of plates you’d like to pour. For example: Because we’d like to make 20 plates, and our plates...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... you should create a 1000x stock solution. For example: If you’ll be preparing plates with a final concentration...

... fresh complete medium. This 5 cells/mL solution will be used to seed the 96-well plate. Sample Calculation...the attachment of adherent cells) Trypsin EDTA (TrypLE, Thermo Fisher 12605-010) 6-well tissue culture...Corning 430167 (optional) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...performed at Addgene on the A549 cells described in our sample data , each 96-well plate gave rise to 15–20 monoclonal...Each 10 cm dish should be seeded in 10 mL DMEM complete, which will generate enough conditioned medium...adding 10 µL homogenized cell solution to 990 µL complete medium. Instead of transferring 0.125 µL, now ...transgene expression and/or other phenotypes. For example, perform Western blotting to screen for lines with...

... 15 mL DMEM Complete. Wednesday: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM Complete. Friday: Plate...your insert of interest Reagent Preparation DMEM Complete (also known as D10): 10% v/v FBS and 4 mM L-alanyl-L-glutamine...Plate 8×10 5 cells in a T75 flask in 15 mL DMEM Complete. Do not add pen-strep to the media. Use cells ...packaging cells at 3.8×10 6 cells per plate in DMEM Complete in 10 cm tissue culture plates. Incubate the cells...at RT. During the incubation, add 10 mL of DMEM Complete to a 15 mL conical and an appropriate amount of...the media. Replace the media with 10 mL of DMEM Complete or OptiPro SFM. Incubate the cells. Virus can ... °C as soon as possible to avoid loss of titer. Sample Data Figure 1: 293T cells were transfected with...

...and valproic acid supplementation During the 24–144 h post-transfection, supplement the flask with 4% ...96 h post-transfection, supplement the flask with 3.75 mM valproic acid. Example feeding schedule: Thursday...similar outcomes when using these protocols. However, please be aware that your protocol may need to be adjusted... HEK293 cells are considered biosafety level 2. Please ensure that you are in compliance with your institution...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 ...

...common to multiple cloning sites, will result in similar sized bands, thus making this simple digest less...Plasmid Size -OR- Insert and Backbone Size The simplest form of diagnostic digest is one in which you ... verification, such as DNA sequencing . In the example above, digestion with enzyme RE1 will linearize...enzyme or enzymes that will cut your plasmid into multiple fragments, you can get a very unique pattern that...patterns so that you get double confirmation. In the example above, digestion with either RE3 or RE4 will give...the insert is in the correct orientation. In the example below we want to know how to differentiate between...

...developed a novel method for the easy assembly of multiple linear DNA fragments (Gibson et al. , 2009). Regardless...Regardless of fragment length or end compatibility, multiple overlapping DNA fragments can be joined in a single...overhangs by chewing back from the DNA 5’ end. Complementary DNA fragments can subsequently anneal to each... DNA Ligase - covalently joins the annealed complementary DNA fragments, removing any nicks and creating...too short to make its own part (<150 bp). Pro-Tip Please note that the way to design the “stitching” primers.... Number of fragments assembled simultaneously Multiple fragments can be assembled in one reaction. However...opens in a new window) Rabe BA, Cepko C. (2020). A Simple Enhancement for Gibson Isothermal Assembly. bioRxiv...

...Add your sample. Spin at 3500 rpm for 5–8 min at 4 °C, discard the flow through. Add more sample and spin...exchange and concentration Note: Both steps could be completed in one (long) day. Equipment Ultracentrifuge T70i...discard the flow through. Repeat this step as needed. Please note that iodixanol is not easily removed. After...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...opens in a new window) copyright (2006). Figure 3: Example of an SDS-PAGE gel of 10 consecutive gradient fractions...

...Mulitchannel Pipetting Technique Learn how to pipette multiple samples at once using a multichannel pipette and avoid...protocol material, visit our Addgene Protocols page . Please feel free to email us at [email protected] with ... avoid inconsistent pipetting with our simple tips Bacterial Transformations We walk you through a standard...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...

...DMEM complete containing 25 µM chloroquine diphosphate and incubate ~5 h For 10 mL of DMEM complete, add... of DMEM complete. Incubate the cells 24–48 h before checking for protein expression. Sample Data Legend...Syringes for filtering Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...HEK293T cells at 3.8x10 6 cells per plate in DMEM complete in 10 cm tissue culture plates. Incubate the cells...

... unknown sample (don’t forget to factor in that dilution when calculating the original sample concentration...similar outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted...science. General Considerations The ELISA can be completed in a single day or broken up over several days...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...

...stretch of DNA to a plasmid, such as: Changing a Multiple Cloning Site (MCS) Adding short tags - ex: HA ...sites and add them to your existing vector. It is a couple days of work that will pay off for years to come...bp We also need to include additional bases to complement the overhangs generated when digesting the vector...could leave off the 3’ G on each oligo (and the complementary C of the other oligo), but this would destroy... competent bacteria and plate. Be sure to pick multiple colonies for mini-prepping and verify insert by...

... one of the four nucleotide bases. This is an example of a trace file from a high-quality portion of ...that these base calls are unreliable. This is an example of a trace file from a portion of a sequencing ...contact Addgene about the accuracy of your plasmid, please email [email protected] and include: The sequencing...trace file. If the mutation is not an artifact, please email [email protected] with the sequence, trace... you can see that just after base 70 there are multiple peaks in the same location. Looking at the trace...

...that don’t cause disease in healthy humans, for example, non-pathogenic E . coli . BSL-2 is for labs that...your workspace with appropriate materials. For example, you may use 70% ethanol, quaternary ammonium, ...pathogens or chemicals that present a danger to people and the environment in biohazard containers. Biohazardous...containers that can be autoclaved. Conclusion Although simple, following appropriate BSL-1 and BSL-2 protocol...reuse or repurpose this SOP in another location, please note that you do so at your own risk; you should...

...is physically attached to the backbone and the complete plasmid can be transformed into bacterial cells...overhanging base pairs on the vector and insert are complementary, the two pieces of DNA connect and ultimately...ultimately are fused by the ligation reaction. The example below depicts the ligation of two sticky ends that... will allow you to verify that the vector was completely digested and if phosphatase treated, that the...phosphatase treatment worked. This control should, in principle, be free of colonies, but the reality is that ...

...similar outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted... the host species of the primary antibody. For example, use an anti-mouse secondary antibody for primary...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...

...between samples. In addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each...location in your recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere ...Annealed-oligo Cloning . If you are lucky enough to have multiple options for enzymes that flank your insert and...gel purification step, it is important to digest plenty of starting material. We recommend 1.5-2μg of donor...

...transforming with very small amounts of DNA or if you're multiple plasmids at once. To save money, many labs also...their own competent cells. This is a relatively simple procedure and is useful for performing low efficiency...so when higher efficiency is needed follow the complete protocol. Thaw the competent cells in your hand...outgrowth (for Ampicillin resistance it is ok to completely skip the outgrowth, for the other antibiotics...

...different samples without cross contamination as long as the tip is changed between samples. Tips can ...pipette ready, push down the plunger. There will be multiple “stops” on the plunger, where you feel resistance... you may consider using a multichannel pipette. Please watch Addgene’s protocol on multichannel pipetting...