...step heats the double stranded DNA template strand to the point where the strands start denaturing and ...range to anneal to each of the single stranded DNA template strands. The DNA polymerase is also stable enough...of the sense and anti-sense strands of the template DNA. They are strands of nucleic acid that are starting...molar ratio. It also requires two unique single stranded DNA oligonucleotide (oligo) primers, which anneal...seconds at 94°C: Continued denaturation of double stranded DNA. Anneal primers for 30 seconds at 55°C: The...polymerase to synthesize and elongate the new target DNA strand accurately and rapidly. Repeat steps 2-4 25-30 ...fill-in any protruding ends of the newly synthesized strands. Materials List Reagents for each 50µL PCR: Thin-walled...

... Day 1: Transform, recover, set up overnight growth (Estimated time 2-3 hours) Transformation should be...of 5 mL (3 mL SOC + 2 mL transformed Endura from two separate transformations). Pro-Tip Do not pipette...assumes familiarity with standard bacterial transformation and basic knowledge of the library being amplified...amplified. Please see our protocol on Bacterial Transformation . The generalized pooled library amplification...60242-1) Alternatives include Stbl4 cells or other ultra-high efficiency electrocompetent cells that are ...using a new vented falcon tube for every two transformations. Each of the four 14 mL Vented Falcon Tubes...repeatedly or mix when removing SOC containing transformed DNA-Endura from cuvette. Shake four 14 mL Vented...

...the trace file, you will see that these base calls are unreliable. This is an example of a trace file...with the sequence, trace file, and primer used. What program can I use to view my trace file? There are ...You should receive your sequencing results as a trace file (.ab1) which graphically depicts the sequence...four nucleotide bases. This is an example of a trace file from a high-quality portion of a sequencing...and include: The sequencing result The original trace file (.ab1 file) provided by your sequencing company...sequencing result, what should I do? Check your trace file first; the apparent mismatch/mutation may be... be the result of a mis-called peak in the trace file. If the mutation is not an artifact, please email...

...the DNA concentration and purity will vary. For more information on determining DNA concentration and purity... that digests single-stranded RNA. Optional: Perform phenol-chloroform extraction - see protocol below...Bacterial Culture You may also like... Bacterial Transformation Agarose Gel Electrophoresis Agarose Gel DNA...biology techniques require highly purified and concentrated plasmid DNA. This page will discuss the general...micrograms to as much as several miligrams and at concentrations ranging from 150 ng/μL to several μg/μL. The...optional) Reagents Overnight culture of bacteria transformed with your plasmid Resuspension buffer Denaturing...elute the DNA off of the column using water or a neutral buffer such as TE. You will now have plasmid DNA...

...Dilute or concentrate purified recombinant antibody to a concentration of 0.9–1.1 mg/mL. Transfer 5 µL of...Lab protocol to determine purity and concentration of recombinant antibodies using ready-to-use biosafe...Collection Antibody Blog Posts Antibody Guide Transfection for Recombinant Antibodies Recombinant Antibody...protocol describes how to determine the purity and concentration of recombinant antibodies using ready-to-use...serial dilutions of a standard antibody of known concentration. After staining with Coomassie blue, protein... Inclusion of this information is solely for transparency intended to support reproducibility in science... video to learn how to determine purity and concentration of recombinant antibodies using Coomassie stain...

... tips Bacterial Transformations We walk you through a standard plasmid transformation protocol and offer...tricks to reduce transformation time and increase efficiency Bacterial Transformation Protocol Inoculating...cells AAV Purification by Iodixanol Gradient Ultracentrifugation Protocol Mulitchannel Pipetting Technique...biology or cell biology. Learn some of the key strategies of aseptic technique, including personal protective... and maintaining sterility while working. AAV Titration by qPCR Use qPCR to measure the physical titer... your adeno-associated virus preparation. AAV Titration by qPCR Using SYBR Green Technology Protocol Agarose...when requesting samples from Addgene Technology Transfer How to Order A step-by-step walkthrough of Addgene's...

...cells transduced F = Fraction of cells with fluorescence D = Dilution Factor V T = Transduction Volume... were transduced with a range of dilutions of pHAGE-TO-dCas9-3XmCherry . 72 h post transduction, cells...Workflow Timeline Day 0: Seed 293T cells Day 1: Transduce cells Day 2 (am): Remove media, replace with fresh...brands and lots of FBS can promote or inhibit transfection. Test a variety of brands and lots of FBS to...to the media, as it can affect viability and transduction efficiency. Titer will vary between cell lines...1468.5 1.5 1:75 20 1478.5 1.5 1:100 15 1483.5 1.5 Untransduced 0 1498.5 1.5 Mix the dilutions well Gently aspirate... one dilution with one well remaining as the untransduced control). Count the cells in the remaining well...

... the absorbance/transmission of light through a liquid to determine the concentration of a particular ...the concentration of a substance in that liquid. In order to accurately measure the concentration of a...significantly in concentration. DNA dissolved in water is going to have a greater variance in concentration readings...Calculate your DNA sample concentration and purity using Addgene’s DNA Quantification Protocol. Protocols... to get a quick and accurate reading of DNA concentration and purity. This can be done with a spectrophotometer...spectrophotometer that can accurately read DNA concentration and purity in as little as 1μl. Regardless of...lid and click measure, be sure to record the concentration and purity. Note: Purity is measured under the...

...time-consuming ligation step of traditional cloning methods. In traditional cloning, base-pairing in the ...reactions by holding the vector concentration fixed across multiple concentrations of insert, thereby increasing...now ready for transformation. Use 1-2 μl of annealing reaction for each transformation, following a standard...stability to hold the plasmid together through the transformation/replication process. LIC employs long overhangs...association between fragments, allowing for transformation without ligation. Because of its dual polymerase...same principle (homologous to the first G on 3'-5' strand from cut site). For simplicity, only the 5' primer...instead of TE buffer) to ensure optimal salt concentrations in subsequent reactions. Step 3: Create Vector...

...to perform gel electrophoresis. Equipment Casting tray Well combs Voltage source Gel box UV light source...recipe here ) Agarose Ethidum bromide (stock concentration of 10 mg/mL) Procedure Pouring a Standard 1%.... Pro-Tip Agarose gels are commonly used in concentrations of 0.7% to 2% depending on the size of bands... given volume to make gels of other agarose concentrations (e.g., 2 g of agarose in 100 mL of TAE will...Optional : Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about...DNA and allows you to visualize the DNA under ultraviolet (UV) light. Caution EtBr is a known mutagen. ...you can image the gel. Pour the agarose into a gel tray with the well comb in place. Pro-Tip Pour slowly...

...plasmid backbone. Transformation Transform your ligation reaction into your bacterial strain of choice. Follow...from your transformation will give you the first indication as to whether your transformation worked. our...Digest of Plasmid DNA DNA Ligation Bacterial Transformation Summary The following technique can be used...purification method, it is important to determine the concentration of recovered DNA . Ligate your insert into your...is difficult to calculate this based on DNA concentration alone. One method is to conduct 2 ligations...competent cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent...conduct a positive control to ensure that your transformation worked. You should also verify that you are...

...recipient plasmid backbone. Transformation Proceed with the transformation according to the manufacturer...from your transformation will give you the first indication as to whether your transformation worked. Your...Digest of Plasmid DNA DNA Ligation Bacterial Transformation Summary PCR based cloning is incredibly versatile... molecular cloning consist of: Leader Sequence: Extra base pairs on the 5' end of the primer assist with...from the gel, it is important to determine the concentration of recovered DNA . Ligate Your Insert Into Your...is difficult to calculate this based on DNA concentration alone. One method is to conduct 2 ligations ...competent cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent...

...Restriction Digest of Plasmid DNA Bacterial Transformation Background Information The final step in the...the backbone and the complete plasmid can be transformed into bacterial cells for propagation. The majority...recognition sequence, which results in a single stranded overhang on the digested end of the DNA fragment...vary depending on the size of each and their concentration. However, for most standard cloning (where the... H 2 O to a total of 10μL Notes: If the DNA concentrations are low such that you cannot get all 100ng ...ligation reactions, especially if using "high concentration" ligase, 5min at room temperature is enough.... incubation at 37°C. Proceed with bacterial transformation . Tips and FAQ Do controls When doing ligations...

...desired concentration in antibody dilution buffer. Pro-Tip The optimal antibody concentration will vary... Inclusion of this information is solely for transparency intended to support reproducibility in science...specific to your antibodies, such as antibody concentrations, incubation times, and recommended compatible...conditions for your target. The optimal antibody concentration will vary between antibodies. Review the manufacturer's... before starting your experiment and consider titrating your antibody to determine the optimal dose. To...you know expresses the protein, such as cells transfected with a plasmid to express the protein of interest...

...of a Gibson Assembly is a fully ligated double-stranded DNA molecule. This has proven to be an efficient...′ end that anneals to the target sequence. One strategy is to order primers that are 60 bp long, with ...antibiotic resistance gene to effectively create an extra part, one part has half of the antibiotic gene and... the DNA fragments are present in equimolar concentrations. The Gibson Assembly master mix (isothermal...for the reaction: T5 Exonuclease - creates single-strand DNA 3’ overhangs by chewing back from the DNA 5...fragment. Pro-Tip Add Extreme Thermostable Single-Stranded DNA-Binding protein (ET SSB) to the isothermal...You can purchase master mix or make your own. Transform bacteria with the DNA and screen for the correct...

... DNA . Antibiotic Concentrations Commonly Used Antibiotics Recommended Concentration Ampicillin 100 µg... glass bottle: 4 g NaCl 4 g Tryptone 2 g Yeast Extract and dH 2 O to 400 mL Note: If your lab has pre-...add the appropriate antibiotic to the correct concentration ( see table below ). Note: If you intend to ...in a shaking incubator. Note: Some plasmids or strains require growth at 30°C. If so, you will likely ...mL Kanamycin 50 µg/mL Spectinomycin 50 µg/mL Tetracycline 10 µg/mL Tips and FAQ What is the difference...

...light path . Light travels from the light source through a condenser that helps concentrate the light onto...powerful (but are cheap and accessible) to massive transmission electron microscopes that allow us to see cellular...and reflect the light. These differences create contrast , which allows you to distinguish objects within...within the sample. You can increase contrast by a) adjusting the aperture of the condenser diaphragm to ...The light that does pass through the sample then travels through the objective lens which magnifies the ...

...especially for your eyes!), remove it from any gel tray as plastic will block much of the UV and with a ...on a glass plate if available. Unlike the plastic tray, this will not significantly reduce the UV, but ...with an empty tube. Alternatively, you can just subtract the weight of the empty tube from the weight of...the (Link opens in a new window) QIAquick Gel Extraction Kit . Always follow the manufacturer's instructions...Note: It is usually important to determine the concentration of the DNA that you isolated before proceeding...

...cross-contamination with other substances. They also help you transfer the material to your tube or container. Pro-Tip...you weighed out too much material, don’t put any extra back into the original container. Dispose of the...Pro-Tip When you weigh out a reagent, you may leave trace amounts of it on the balance. Therefore, it’s best...weighed out the correct amount of your reagent, transfer it into your container. Make sure that any spills...Discard the weighing boat or weighing paper in the trash if the material weighed is not biohazardous. If ...

... that you have received hazardous waste safety training before starting the work. Before working with ...needles. Sharps containers must be thick walled, impenetrable by a needle, and must be able to close securely... safety protocols require bloodborne pathogens training. It is strongly recommended that anyone participating...BSL-2 waste can be decontaminated in a final concentration of 10% bleach for 30 minutes before pouring ...adhered to. None of the authors, contributors, administrators, or anyone else associated with Addgene, can...