Column clogging can result from the following:

• The sample is too large.

  Too many cells were applied to the column. Ensure that the amount of cells used is less than 2 x 10⁹ viable cells, and that no more than 1 mL of culture is applied to the column/well. Clogging can be alleviated by increasing the g force and/or centrifuging for a longer period of time until the lysate passes through the column.

• Insufficient Vacuum (for High-throughput only).

  Ensure that a vacuum pressure of at least -650 mbar or -25 in. Hg is developed.

• Centrifuge temperature is too low.

  Ensure that the centrifuge remains at room temperature throughout the procedure. Temperatures below 15℃ may cause precipitates to form that can cause the wells to clog.

Experiencing gelatinous lysate prior to loading onto the column may be due to:

• The lysate/binding solution mixture is not homogeneous.

  To ensure a homogeneous solution, vortex for 10-15 seconds before applying the lysate to the spin column.

• The sample is too large.

  Too many cells are in the lysate preparation. Ensure that the amount of cells used is less than 2 x 10⁹ viable cells, and that no more than 1 mL of culture is applied to the column/well.

A low genomic DNA yield may be caused by the following:

• The sample is old/overgrown.

  The culture may have been overgrown, allowing lysis of older cells to occur more readily. This will lead to premature degradation of the genomic DNA. It may be necessary to use bacterial cultures before they reach maximum density.

• Incomplete lysis of cells.

  Extend the incubation time of Proteinase K digestion or reduce the amount of bacterial cells used for lysis. Increase the lysozyme incubation time for gram-positive strains.

• The DNA elution is incomplete.

  Ensure that centrifugation at 20,000 x g is performed after the 3,000 x g centrifugation cycle to ensure that all the DNA is eluted.

Sheared genomic DNA could be caused by:

• Improper handling of genomic DNA.

  Pipetting steps should be handled as gently as possible. Reduce vortexing times during mixing steps (no more than 10-15 seconds).

• The cells are old.

  Older cultures contain prematurely lysed cells which release endonucleases and can degrade DNA. Fresh cultures are recommended.

If the DNA does not perform well in downstream applications, it may be due to one or more of the following:

• DNA was not washed two times with the provided Wash Solution A.

  Ensure the column/plate was washed two times with Wash Solution A. An additional wash with Wash Solution A can improve DNA performance in downstream applications, although it may reduce DNA yield.

• Ethanol carryover.

  Ensure that the column/plate drying step after the Wash procedure is performed in order to remove traces of ethanol prior to elution. Ethanol is known to interfere with many downstream applications.

Yes, the DNA from this kit is good for both Nanopore sequencing and Long-read sequencing. Please contact our Tech support team at support@norgenbiotek.com and ask for reference publications.