pcr reaction cleanup buffer
A buffer and polyol technology, applied in biochemical equipment and methods, DNA preparation, microbial determination/inspection, etc., can solve problems such as difficult to wash off
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Embodiment 1
[0072] Example 1: Preparation of Magnetic Particles and Binding Buffer
[0073]Experimental DNA binding buffer To standard 20 wt% PEG 8,000mw dissolved in 2.5 molar sodium chloride solution was added several Perstorp polyols, "Alkoxylated Pentaerythritol", as the DNA binding buffer (PEG / salt binding buffer Agents can be described, for example, in U.S. Patent No. 5,705,628, and Solid-phase reversible immobilization for the isolation of PCR products ("solid-phase reversible immobilization for separating PCR products"), MargaretM.DeAngelis, David G.Wang and Trevor L. Hawkins, Nucleic Acids Research, 1995, Vol. 23, No. 22, pp. 4742-4743). The experimental buffer system currently employs 2.5 molar NaCl and 10-40% Perstorp polyols (e.g., polyol 4290, polyol 4840, polyol 4640, polyol R3215, and polyol R6405) as water grabbing / Water attracting additives. The binding buffer mixture also contained 10 mM tris and 1 mM EDTA. Commercially available beads (Acegen Corporation, Foster C...
Embodiment 2
[0074] Example 2: PCR reaction and cleanup protocol
[0075] Using standard PCR kits, two PCR reactions were used to generate 200bp and 700bp fragments. The two PCR reactions were mixed 1:1 (v / v) to generate a PCR mixture containing both 200bp and 700bp fragments. The following protocol was used for all clearance evaluations: (1) a total of 20 μl of crude PCR solution was pipetted into a 1 ml tube, then 36 μl of magnetic bead / particle solution was added and mixed; (2) after 5 minutes of binding, the beads / particle The solution is placed on the magnet for 2-5 minutes, and the supernatant is collected with a pipette and discarded; (3) when the beads / particles are still on the magnet, wash with 70% alcohol solution (200ul each) Twice, then discard the alcohol eluate; (4) Elute the captured DNA by adding 40 μl of 1×TE buffer.
Embodiment 3
[0076] Example 3: UPLC analysis of eluted DNA fragments
[0077] DNA eluted from magnetic beads / particles was identified and quantified by UPLC analysis on a C18 reverse phase column using a Waters UPLC system. Both 200bp and 700bp DNA fragments were detected and quantified under 260nm ultraviolet light using a Waters PDA detector. 10 μl of the eluted DNA solution of each sample was injected for comparison. Yields of eluted DNA were based on peak areas of 200 bp and 700 bp DNA fragments. The remaining DNA primers used in this reaction were also detected using UPLC, but no primers were observed after cleanup.
[0078] A more detailed consideration of the characteristics of buffers for magnetic affinity-bonded thermoplastic solid-phase extraction, Figure 1-4 The graph shows binding buffer performance, which shows the release using magnetic particle separation for experimental solutions B1, B2, B3, B4, B5 and B6 (as defined in Table 1), Axygen buffer and PEG buffer. DNA (m...
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