...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...Transfection grade DNAs 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...