...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...

...desired bacterial strain and you will not need to obtain more competent cells and retransform. Bacteria on ... of Plasmids You may also like... Bacterial Transformation Recovering Plasmid DNA from Bacterial Culture...the following day. Tips and FAQ The optimal concentration of long-term glycerol storage is unknown. Most...

...Digest of Plasmid DNA DNA Ligation Bacterial Transformation Summary Oligo overlap cloning can be used anytime...50mM NaCL, 1mM EDTA) and mixed in equimolar concentrations. We recommend mixing 2μg each in a total volume...with 45μL nuclease-free water and quantify the concentration (should be about 8ng/μl). Mix the annealed oligos...the vector with 0.75-6 ng of annealed oligos). Transform 2-3μL into your favorite competent bacteria and...

...complementary to template strand). However, primers do not need to correspond to the template strand completely; it...primer corresponds completely to the template DNA strand so elongation can proceed. Usually a guanine or...

...These items should remain in the laboratory. When traveling between lab spaces, take your glove off of one... Consider nitrile gloves as an alternative. For extra precautions, tuck your sleeves into your gloves ...adhered to. None of the authors, contributors, administrators, or anyone else associated with Addgene, can...

...experimental procedures, such as minipreps and RNA extractions. Plus, centrifuges are useful for simply collecting...cousins, but can hold much larger containers. Ultracentrifuges are also generally floor sized centrifuges...you may need to add a blank. Blanks are often an extra tube filled with water to the same volume as your...

...Plasmids grown in Dam or Dcm methylase positive strains will be resistant to cleavage at certain restriction...variety of reasons, including high glycerol concentration. Learn more at (Link opens in a new window) ...

...also like... Making LB Agar Plates Bacterial Transformation Recovering Plasmid DNA from Bacterial Culture...

...digest. Although this is never an ideal cloning strategy, sometimes it cannot be avoided. If you do have...