... that has UV light, visualize your DNA fragments. The fragments of DNA are usually referred to as ‘bands...DNA by size (e.g., length in base pairs) for visualization and purification. Electrophoresis uses an electrical.... Pro-Tip TBE can be used instead of TAE, labs usually use one or the other, but there is very little ... concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel...gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light. Caution ...loading less DNA into the well. Another method for visualizing very short DNA fragments is polyacrylamide gel...

...digestion (usually 3-6bp). Restriction Site: Your chosen restriction site for cloning (usually 6-8bp). Hybridization...primer that binds to the sequence to be amplified (usually 18-21bp). When selecting restriction sites, you...the desired location in your recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not...genomic DNA or a cDNA library), roughly 18-21bp is usually sufficient to give specificity and to also be compatible... run the product on a gel. This allows you to visualize that your PCR product is the anticipated size ...

...enzyme is usually better. Mix gently by pipetting. Incubate tube at appropriate temperature (usually 37 °C...requires 1 µg of DNA. The total reaction volume usually varies from 10-50 µL depending on application and...manufacturer's instructions for more details. To visualize the results of your digest, conduct gel electrophoresis...

...transformation efficiency. The lowest efficiency cells (usually the least expensive) are fine for transforming ...incubate in 37°C incubator. Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 20-50 μL of competent cells...into a 42°C water bath for 30-60 secs (45 secs is usually ideal, but this varies depending on the competent...

...organism's genome, for example. Amplification is usually necessary to produce sufficient quantities of library...the shared plasmid backbone a single time and visualize that digestion on an agarose gel ( see restriction...all liquid has been absorbed by the agar. This usually takes 1-2 minutes. Critical Be careful not to rip...

...proceed. Usually a guanine or cytosine is used at the 3’ end, and the 5’ end of the primer usually has stretches...

...Note: If you intend to do a mini-prep you will usually want to start 2 mL in a falcon tube, but for larger...within a single bacterial cell. Large plasmids usually have a low copy number (approximately one or two...

...sticky ends that were generated by EcoRI digestion: Usually, scientists select two different enzymes for adding...Ratio Although a 3:1 insert to vector ratio is usually sufficient, you can optimize the amount of insert...

... bacteria with resistance to that antibiotic - usually conferred by a plasmid carrying the antibiotic ...H 2 O Sterile plates of your desired size - we usually use 60 mm x 15 mm plates which can hold 5-10 mL...

...follow the manufacturer's instructions. Note: It is usually important to determine the concentration of the...

...base-pairing in the short overlapping regions (usually 4 bp) does not provide enough stability to hold...

...laboratory procedure for separating DNA by size for visualization and purification. Agarose Gel Electrophoresis...

...the dilutions well Note: the 1:10 dilution can usually be omitted because this dilution typically produces...

...the desired location in your recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not...

...of a second peak at a temperature of ~70–75 °C usually indicates the presence of primer dimers which can...

...video to learn how to use western blotting to visualize a protein from cells or tissue samples. Equipment...

...in a sterile microcentrifuge tube. TIP: When visualizing DNA fragments to be used for ligation, use only...