Method for purifying biomolecules
A technology of biomolecules and biological samples, which is applied in the field of nucleic acid purification such as DNA and RNA molecules, and can solve problems such as insufficient yield
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Embodiment 1
[0079] Example 1: Basic procedure (single-step method as described in the prior art)
[0080] Colonies growing on agar plates and containing the plasmid to be isolated were picked, 3 ml of LB liquid medium per suspension, and proliferated by overnight incubation at 37°C. 3 ml of the saturated bacterial overnight culture was pelleted by centrifugation at 13000 rpm in a tabletop centrifuge. Plasmid DNA was isolated by Qiagen's modified routine protocol according to Birnboim's method. Remove and discard the bacterial culture supernatant. 250 μl of P1 buffer (Qiagen) was added to the pellet and the pellet was resuspended. Bacteria were lysed (alkaline lysis) by adding 250 μl of P2 buffer (Qiagen) and shaking carefully 4-5 times; the lysis reaction should not last longer than 5 minutes, otherwise the genomic DNA will become active. Therefore, the lysis reaction was stopped by adding 350 μl of N3 buffer (Qiagen) and immediately with gentle shaking. Lysed bacterial cell wall comp...
Embodiment 2A
[0082] Example 2A. Comparison of DNA yields between single-step and two-step centrifugation methods (combined steps)
[0083] 3 ml of bacterial culture (DH10B) containing plasmid puc 19 were collected and lysed as described above and transferred to spin columns (QIAprep type), followed by conventional one-step ("artificial one-step protocol") or two-step ("artificial one-step protocol"). Manual two-step combination") centrifugation. The process parameters are as follows:
[0084]
[0085] The main differences in the centrifugation protocol have a grey background. Buffers P1, P2, N2, PE and EB are components of the QIAprep kit. The yield of plasmid DNA was then investigated. Eight parallel experiments were performed in each case and the results were statistically evaluated and presented in Figure 2A middle. While a DNA yield of 8454 ng was achieved with the single-step method, a yield of 9540 ng was achieved with the two-step method. The difference is significant. It...
Embodiment 2B
[0086] Example 2B. Comparison of DNA yields between single-step and two-step centrifugation methods (washing steps)
[0087] Similar differences were found when the washing step was designed as two stages instead of the binding step, as shown in the table below:
[0088]
[0089] The main differences in the centrifugation protocol have a grey background. Eight parallel experiments were performed in each case and evaluated as in the previous example. The results are displayed in Figure 2B middle. While a DNA yield of 4022 ng was achieved with the single-step method, a yield of 4803 ng was achieved with the two-step method. The difference is significant. It can be clearly seen that the DNA yield is about 19% higher with the two-step method.
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