Conventional PCR using agarose gel electrophoresis detection Essay

Conventional PCR using agarose gel electrophoresis detection - Essay Example While addition of gel, the care for the percentage of it has to be taken as â€Å"a 0.7% gel will show good separation (resolution) of large DNA fragments (5–10kb) and a 2% gel will show good resolution for small fragments (0.2–1kb).† So, the percentage of the gel is kept between 0.7% to 2%. With intention to separate very tiny fragments, addition of high percentage ( up to 3%), is not useful as a vertical polyacrylamide gel is more appropriate in this case. The medium percentage is always recommended as low percentage gel may break while trying to lift them and high percentage gels may often brittle not setting evenly. Lewis recommends 1% gel to use. While suggesting for gel tank Lewis recommends, â€Å"Small 8x10cm gels (minigels) are very popular and give good photographs.† For the applications of Southern and Northern blotting, larger gels are used. 30–50mL and 205 mL of agarose is required for minigel and larger gel respectively. While deciding the amount of DNA to be added to this solution, the nature of analysis has to be kept in mind. According to Lewis â€Å"Typically, a band is easily visible if it contains about 20ng of DNA.† After doing all the above preparation Lewis says, â€Å"I usually digest and load 2–4 µL of the 50 µL obtained from a kit miniprep. But you see how it depends on the number and size of the bands expected. For PCR reactions, it depends on the PCR but in routine applications 10–20 µL should be plenty to see the product on the gel.† Depending on the volume of DNA being loaded and the number of samples, the design of comb is decided to include in the process. Lewis recommends, â€Å"Combs with many tiny teeth may hold 10 µL. This is no good if you want to load 20 µL of restriction digest plus 5 µL of loading buffer. When deciding whether a comb has enough teeth, remember that you need to load at least one marker lane, preferably two.† After