Editing Plasmid MaxiPrep HIC (section)

===  ===

'''Precipitation of RNA using Ammonium Acetate'''





1. Spin down 500 ml cells

2. Add 20 ml of P1 (50 mM Tris-HCl, 10 mM EDTA pH8.0) without RNase.

3. Add 20 ml of P2 (200 mm NaOH, 1% SDS solution), lyse for about 5 min.

4. Add 20 ml of P3 (7M&nbsp;<span style="line-height: 20.8px;">ammonium acetate</span>).

<span style="line-height: 20.8px;">5. Incubation at 4 degrees overnight</span>

The high salt precipitation may involve adding 1M potassium acetate and 7M ammonium acetate (concentrations of both may be varied, with higher concentrations of ammonium acetate being better for RNA precipitation) and holding the temperature at 4 degrees Celsius for approximately 6 to 12 hours to precipitate RNA (times and temperatures may be varied; lower temperatures and longer times improve precipitation and incubation at 4 degrees Celsius provides a higher percentage of precipitation). The ability to precipitate RNA during neutralization of the alkaline lysate helps eliminate the need for RNase. The acetate solution does not need to be pH adjusted and the pH is approximately 8. Conventional techniques using 3M potassium acetate at pH 5 are dramatically less effective at RNA precipitation and provide greater risk of DNA damage.

<span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">The phenyl 650-S resin (100 mL) was packed into a 5 cm diameter column to a height of 5 cm. The column was equilibrated with approximately 500 mL of 1.75M (NH</span><sub style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; line-height: 21.3333px;">4</sub><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">)</span><sub style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; line-height: 21.3333px;">2</sub><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">SO</span><sub style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; line-height: 21.3333px;">4&nbsp;</sub><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">at 20 mL/minute. Then 350 mL of the DEAE elution pool containing </span><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">plasmid DNA was diluted with an equal volume of 3.5M (NH</span><sub style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; line-height: 21.3333px;">4</sub><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">)</span><sub style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; line-height: 21.3333px;">2</sub><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">SO</span><sub style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; line-height: 21.3333px;">4&nbsp;</sub><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">which results in a final concentration of 1.75M (NH</span><sub style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; line-height: 21.3333px;">4</sub><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">)</span><sub style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; line-height: 21.3333px;">2</sub><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">SO</span><sub style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; line-height: 21.3333px;">4</sub><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">. The entire 700 mL of plasmid DNA in 1.75M (NH</span><sub style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; line-height: 21.3333px;">4</sub><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">)</span><sub style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; line-height: 21.3333px;">2</sub><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">SO</span><sub style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; line-height: 21.3333px;">4&nbsp;</sub><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">was pumped through the HIC column and the flow through peak which contains the </span><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">plasmid was saved for further processing by ultrafiltration and diafiltration (UF/DF). The bound RNA, chromosomal DNA, denatured plasmid and endotoxins were eluted in a Milli-Q water wash followed by a 70% ethanol (v/v) wash.</span>

<span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;">Plasmid should not bind, genomic DNA, RNA should bind to phenyl 650-S.</span>

<span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;"></span>

<span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px; line-height: 21.3333px;"></span>
[[Category:Protocols]]