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View additional product information for PureLink™ Fast Low-Endotoxin Midi Plasmid Purification Kit - FAQs (A36227, A35892)
10 product FAQs found
对于任何硅膜柱，使用水进行洗脱通常都是可行的。使用缓冲液的优点是吸光度的读值会比较稳定和精确，因为纯水的pH值可能会很低（pH4-5）。
260nm吸收值和280nm吸收值的比值（A260/A280）通常被用来判定样本的纯度。对于DNA而言，理想的比值是1.8，但是可以接受的范围为1.7–1.9。A260/A230也经常被用来确定是否存在污染。DNA的A260/A230理想值为1.8–2.0。DNA的纯度也可以用凝胶电泳检测。对于质粒DNA而言，应出现一条明显的单一条带（可能会出现几条额外条带，代表质粒分子的多种形式）。对于基因组DNA，查看高的平均片段的大小。
For any silica columns, elution with water is generally possible. However, a buffer is preferred for stability and accuracy of absorbance readings, as pure water can have a very low pH (4 - 5).
Endotoxins are typically any cell-associated bacterial toxins that are part of the outer surface of the cell wall of gram-negative bacteria. Endotoxins can influence cell growth, cell differentiation, contractility, and protein expression in mammalian cells. Endotoxins are released during bacterial lysis, and they can subsequently reduce transfection efficiency and protein expression levels. Please review the following article for more information about endotoxins: Butash KA et al. (2000) Reexamination of the effect of endotoxin on cell proliferation and transfection efficiency. Biotechniques 29(3): 610-614, 616, 618-619.
The ratio of absorbance at 260 nm to the absorbance at 280 nm (A260/A280) is typically used to measure purity of the sample. For DNA, the ideal A260/A280 ratio is 1.8, but it can be in the range of 1.7 - 1.9. The A260/A230 ratio is also used to determine if contamination is present. For DNA, the ideal A260/A230 ratio is between 1.8 and 2.0. DNA purity can also be examined by gel analysis. For plasmid DNA, look for a strong, single band (perhaps with a few extra bands representing multimers of the desired molecule). For genomic DNA, look for high average fragment sizes.
We offer the following silica column-based plasmid purification kits for isolation of endotoxin-free or low-endotoxin plasmid DNA:
- PureLink Expi Endotoxin-Free Maxi Plasmid Purification Kit (Cat. No. A31217, A31231, A33073) (<0.1 EU/µg endotoxins):
https://www.thermofisher.com/order/catalog/product/A31217
- PureLink Fast Low-Endotoxin Midi (Cat. No. A35892, A36227) or Maxi (Cat. No. A36228, A35895) Plasmid Purification Kits (<1 EU/µg endotoxins):
https://www.thermofisher.com/order/catalog/product/A35892
https://www.thermofisher.com/order/catalog/product/A36228
Our recommendation is to not exceed the maximum recommended culture volumes specified in the kit manuals; using larger culture volumes means increased cell mass, which will result in reduced plasmid yield and purity.
We recommend using high-copy number plasmids cultured in LB medium to obtain maximal yield and purity of plasmid DNA. You can use low-copy plasmids, but please be aware that the expected yield and purity will be considerably lower.
Avoid using rich media for bacterial cultures. If using a rich medium is necessary, ensure that the recommended cell mass is not exceeded by optimizing the growth time so that the OD600 stays at <4.0, or by processing a smaller culture volume (<100 mL).
We recommend using bacterial cultures at OD600 of 2.0 - 4.0.