PureLink™ HiPure Precipitator Module

There are several reasons lower yields can occur:

-Make sure the binding of the plasmid is being done at RT. Temperature affects the pH of the binding solution.

-Make sure all other solutions are also warmed to RT for optimal yield.

-Verify that the centrifugation immediately following the neutralization step was not done at 4 degrees C. If it was, the supernatant MUST be warmed to RT before loading onto the column.

-The 1:1:1 ratio of buffers (Resuspension/Lysis/Precipitation) has not been kept precisely. A surplus of solution E3 will exceed the optimal salt concentration for DNA binding, thus leading to decreased DNA yield.

-Low-copy number plasmids give lower yields.

-If all of the medium was not removed at the cell harvesting step, the pH of the subsequent step can be affected and reduce yield.

-The cell pellet was not thoroughly resuspended before lysis.

-The purified DNA pellet was overdried after isopropanol precipitation and ethanol wash making it difficult to resuspend the pellet. Air dry only.

-Pellets can be easily lost during the alcohol precipitation and wash steps. It is best to pipette off the alcohol solutions rather than pouring them off, as the pellets tend to be slippery.

-After lysis and neutralization, the lysate is not homogeneous. After addition of solutions (Resuspension/Lysis/Precipitation buffers), cells must be mixed thoroughly by inverting the tubes until a homogeneous phase is obtained. Otherwise, cells will not be lysed completely and the plasmid DNA will stick to bacterial debris. In the case of bigger bacterial cell pellets, more vigorous shaking may be required to achieve homogeneity. However, do NOT vortex, as vortexing will release chromosomal DNA.

The DNA pellet from precipitation with isopropanol is easily dislodged when washing with 70% ethanol. It is best to remove the isopropanol supernatant and the ethanol wash by pipetting. Be careful not to shoot the washing buffer directly onto the pellet. Instead, allow the washing buffer to run over the pellet. Regardless of which manufacturer's miniprep kit you use, washing the pellet can be challenging because it is so small.

The binding capacity of the PureLink HiPure Precipitator is 2 mg.

For best results, we recommend the use of the PureLink HiPure Precipitators with our PureLink HiPure Plasmid Filter Maxiprep/Midiprep Kit or PureLink HiPure Plasmid Maxiprep/Midiprep kit. However, PureLink HiPure Precipitator can be used with an equivalent anion exchange plasmid purification kit. We do offer the PureLink HiPure Precipitator as a stand-alone product (Cat. Nos. K210021 and K210022).

It is not recommended. We cannot guarantee the yield or the quality of the DNA from a re-used Precipitator.