JRD200 plasmid extraction experiment

select method is alkaline cracking method. If there is a problem, or a large number ( u0026 gt; 15 kb), use a mild method SDS lysis method. Details View u0026 ldquo; Molecular Clone u0026 Rdquo ;. Both the kit uses alkaline cracking method, so there is a common problem, and the effect is not good when you extract large plasmid. The common method of plasmid extraction experiment is alkaline cracking method, which has high yield, wide range, rapid, and high purity. Regarding the principle of alkaline cracking method, there is a monolith in Fudan University Studies and Molecular Biology Laboratory. You can go to see. This is an article, very interesting. The article refers to a different point of view that can be found, it is also very inspired.

Alkaline cracking method is a very varied method, the plasmid varies in alkaline conditions, time is long, this denaturation is irreversible (a little bit in front of the super spiral during electrophoresis, if there is a belt, the plasmid of this change .). Therefore, to reduce the inverse degeneration, the time to control the alkali crack. (It seems to be such a reasoning: under alkaline conditions, the two chains of the plasmid are separated from a point or a few points, and over time, until it is separated. In theory, the separate two strands should be very fast. Pairing rehabilitates, no separate two chains may be paired.) The alkali cracking method is not suitable for the fact that the reason is too violent, so that the proportion of super screw is lower. The method recommended by the literature is a much gentle method, and the disadvantage is that the rate is low. The problem now is that the number of copies of large plasmids is low. If the extraction method is not high, it is very laborious. If it is noted that in the alkali cracking method, the super-spiral ratio decreases as the alkali cleave time, with this phenomenon as the viscosity increases, the alkaline cracking method can be used to extract large plasmid: increase the use of the reagent After adding NaOH / SDS solution, the solution can become very clear within 1 minute; immediately add neutralization reagent. We did not have done this experiment, but qiagen saves a large number of alkali solution.