Paul.xu at 16:07, 28 July 2017

2017-07-28T16:07:10Z

New page

P1 without RNase

P2 is the same

'''Modified N3:'''

N3 with 2.5 M CaCl2 (for removing RNA), 30 mM MgCl2 (for precipitating with PEG 8000)

1. 0.25 ml P1

2. 0.25 ml P2

3. 0.35 ~ 0.5 ml N3,

or 0.9 ml  modified N3  (0.36 ml N3 + 0.55 ml 3.3 M CaCl2, final conc of CaCl2 is 2 M)

After adding modified N3, CaCl2 conc is 1~1.3 M

4. Spin

5. Collect supernatant

7. Precipitate with isopropanol (1 vol)

or 0.3 vol 40% PEG 8000, 1 M NaCl2

8. Wash 2X with  75% ethanol

'''Removal of RNA by CaCl2 and Precipitation with PEG'''

Two salts (LiCl and CaCl2) were tested for selective precipitation of RNA from the plasmid DNA in solution. LiCl is widely used to precipitate high molecular weight RNA [1,3,11]. For removal of RNA by LiCl precipitation, 1 vol of 6 M LiCl was added to the cleared lysate to obtain a final concentration of 3 M LiCl The samples were mixed, left at room temperature for 10 min, and centrifuged at 12,000g for 10 min. The resulting supernatant containing the plasmid DNA was transferred to new tubes. For removal of RNA by CaCl2 precipitation [12], a stock solution of CaCl2 at 4.2 M was added to the cleared lysate to obtain a final concentration of 1.4 M, and the samples were mixed followed by immediate centrifugation at 12,000g for 10 min

After collecting the cleared lysates from the alkaline lysis step, a stock solution of '''4.2 M '''of CaCl2 was added to a final concentration of '''1.4 M'''. The tubes were spun immediately at RT and the supernatants were transferred into a new tube. The nucleic acids were then precipitated by standard isopropanol precipitation (0.3 M sodium acetate, 0.6 vol isopropanol). After '''10''' min at RT, the nucleic acids were pelleted by centrifugation at '''12,000g for 10 min'''. The pellets were washed with 70% ethanol, dried, and resuspended in 50 ll of TE.

An equal volume of 20% PEG, 500 mM NaCl was added to the samples with vigorous mixing to achieve the final concentration of '''10% PEG, 250 mM NaCl'''. Immediately after mixing, the samples were centrifuged at 12,000g at RT for 10 min. The supernatants were discarded and the pellets washed with 70% ethanol, dried, and resuspended in 30 ll of 10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0
[[Category:Protocols]]

RNAse-Free MiniPrep - Revision history

Paul.xu at 16:07, 28 July 2017