Instructions

Note: In order to eliminate endotoxins, use endotoxin free (pyrogen free) water throughout the process and operate in controlled conditions. When using prepacked gravity columns, please skip step 3.1.

Generic column packing instructions

The Seplife® Pd oneS resin is recommended to be packed in a column by gravity. Mix well the resin avoiding using harsh mixing tools or conditions that could damage the particles. Pour the homogenate in the gravity column with the lower distribution plate in place. Fix the upper distribution plate of the column.

Equilibration

Equilibrate the column by gravity flow with 2-5 times the bed volume (BV) of an equilibration buffer such as:  50 mM Tris-HCl, 0.25 M NaCl, 10 mM EDTA, pH 7.5, so that the conductivity and pH of the effluent are consistent with those of the loading buffer.

Sample loading

(1) Sample pretreatment: Prepare the sample using alkaline lysis method. It is recommended to resuspend the bacterial sludge at a ratio of 5:1 (volume to mass ratio), then add 0.2M NaOH and 1% SDS for 4-10 min under slow stirring. During the alkaline lysis process, it is important to control the addition of the alkaline solution and the contact time to avoid irreversible damage to the plasmid and affect the recovery rate of the pDNA. Finally, use 3M potassium acetate pH 5.5 to neutralize the lysate. After standing for 30 minutes , centrifuge the lysate to obtain the supernatant or use membrane filtration to clarify before loading on the column.

(2) Sample loading: Samples are usually prepared and used immediately. It is not recommended to use samples that have been stored for a long time, as this will affect the purification effect.

Washing​

After sample loading, use a wash buffer to remove RNA, endotoxins, HCP and other impurities. To maximize sample purity, it is recommended to use 3-5 BV or higher . Example of wash buffer: 50mM Tris-HCl, 0.45 M NaCl, 10mM EDTA, pH 7.5.

Elution

(1) Example of elution buffer: 50mM Tris-HCl, 1.25M NaCl, 10mM EDTA, 15% isopropanol, pH 8.5. It is recommended to use 1-2 BV of elution buffer to ensure an efficient recovery of plasmid DNA.

(2) Eluted sample processing: Add isopropanol to the elution fraction to a final concentration greater than 30% to encourage pDNA precipitation. After standing at 4ºC for 30 min, centrifuge at ≥10,000 G for 20 min, and then gently remove the supernatant (no shaking to reduce loss). Quickly add 1/4 of the elution volume of 70% or anhydrous ethanol to wash the pDNA sample. After standing at 4ºC for 30 min, centrifuge at ≥10,000 G for 30 min. Finally, gently remove the supernatant (no shaking to avoid removing solid particles). Completely evaporate the ethanol in the centrifuge tube and add ultrapure water or nucleic acid preservation buffer to re-dissolve the precipitated pDNA and store for later use.

Cleaning in Place (CIP)

After pDNA elution, rinse the gravity column with 3 BV ultrapure water, then use 0.5M NaOH solution to perform CIP in situ by soak it in 2 BV 0.5M NaOH solution for 15-30 minutes, or use 5 BV 0.5M NaOH solution for dynamic cleaning. After the CIP is completed, rinse the resin with ultrapure water to remove all alkaline solution.

Storage

Store in closed containers at 4-30℃, in a dry, ventilated and clean place, away from direct sunlight. Do not freeze. Used gravity columns should be stored at 4-30℃ in 20% ethanol solution.

Ordering information