Method for purifying plasmid DNA
A plasmid, stable technology, applied in the field of plasmid DNA liquid formulations, can solve problems such as degradation reactions affecting DNA stability, increasing the risk of contamination and/or degradation, etc.
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Embodiment 1
[0219] The adjustment of the diameter to the rate used is derived from the calculation of the Reynolds number in the coils of the continuous cracking system. Because the following analysis assumes that the fluid properties are Newtonian, the calculations reported below are only fully valid in B1a and to some extent in B2.
[0220] The value of the Reynolds number allows one skilled in the art to determine the type of characteristic encountered. Here, we will only focus on fluid flow in pipes (hydraulic engineering).
[0221] 1) Non-Newtonian fluid
[0222] The two most commonly encountered non-Newtonian fluids in industry are Bingham and Ostwald de Waele.
[0223] In this case, the Reynolds number (Re) is calculated as follows:
[0224] Re N is the generalized Reynolds number
[0225] Re N =(1 / (2 n-3 ))×(n / 3n+1) n ×((ρ×D n ×w 2-n ) / m) (1)
[0226] D: Inner diameter of cross section (m)
[0227] ρ: Volume mass of fluid (kg / m 3 )
[0228] w: space velocity of the f...
Embodiment 2
[0269] We can decompose the CL system into 5 steps. In a specific embodiment, the configuration is as follows:
[0270] 1) Mixing: cells (in solution 1) + solution 2 (M1 + 3m of 6mm tubing). Cells are initially lysed by SDS, there is no risk of fragmentation as long as the DNA is not denatured.
[0271] 2) End of lysis and denaturation of gDNA (13m of 16mm tubing).
[0272] 3) Mixing: lysate + solution 3 (M2 + 3m of 6mm tubing).
[0273] 4) Harvest the neutralized lysate at 4°C
[0274] 5) Settling flocs and large gDNA fragments overnight at 4°C.
[0275] The following conditions can be used to perform sequential lysis:
[0276] - Solution 1: EDTA 10 mM, Glucose (Glc) 9 g / l and Tris HCl 25 mM, pH 7.2.
[0277] - Solution 2: SDS 1% and NaOH 0.2N.
[0278] - Solution 3: acetic acid 2M and potassium acetate 3M.
[0279] - Flow rate 60 l / h: solution 1 and solution 2
[0280] - Flow rate 90 l / h: solution 3.
[0281] - The cells were adjusted to 38.5 g / l with solution 1. ...
Embodiment 3
[0298] The column used was a 1 ml HiTrap column activated with NHS (N-hydroxysuccinimide, Pharmacia), connected to a peristaltic pump (output 2 group, its sequence is as follows:
[0299] 5'-GAGGCTTCTTCTTCTTCTTCTTCTT-3' (SEQ ID NO: 1)
[0300] The buffers used in this example are as follows:
[0301] Coupling buffer: 0.2M NaHCO 3 , 0.5M NaCl, pH 8.3.
[0302] Buffer A: 0.5M ethanolamine, 0.5M NaCl, pH 8.3.
[0303] Buffer B: 0.1M Acetate, 0.5M NaCl, pH 4.
[0304] The column was washed with 6 ml of 1 mM HCl, then the oligonucleotide diluted in coupling buffer (50 nmol in 1 ml) was applied to the column for 30 minutes at room temperature. The column was washed three times with 6 ml buffer A followed by 6 ml buffer B. The oligonucleotides are thus covalently bound to the column via CONH linkages. Store the column in PBS, 0.1% NaN at 4°C 3 , can be used at least 4 times.
[0305] The following two oligonucleotides were synthesized: oligonucleotide 4817: 5'-GATCCGAAGAAGAAG...
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