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...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...