1. Why is the micro spin column clogged?
    1. Centrifugation speed was too low or spin time was inadequate - Check the centrifuge to ensure that it is capable of generating the require RPMs. Sufficient centrifugal force is required to move the liquid through the resin. Also ensure that the correct spin times are followed. Spin for an additional minute if necessary.
    2. Bacterial debris or residual agarose are still in the lysate - Ensure that the starting material is clarified phage supernatant. Ensure that bacterial debris and residual agarose from the initial phage supernatant is removed by centrifugation at 10,000 × g for 10 minutes before beginning the protocol.
    3. 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.
    4. Centrifuge temperature too low - Ensure that the centrifuge remains at room temperature throughout the procedure. Temperatures below 20°C may cause precipitates to form that can cause the columns to clog.
  2. Why is the yield of genomic DNA low?
    1. Ineffective propagation of phage and initial lysis step - Refer to manufacturers recommendations for the propagation of the phage, including proper titer for inoculation, growth conditions, and bacterial host.
    2. Incomplete lysis of cells - Ensure that the incubation was performed for 15 minutes at 65ºC after the addition of lysis buffer.
    3. Incomplete lysis of cells - Perform optional digestion with Proteinase K in Step 1a during Lysate Preparation.
  3. Why does the DNA not perform well in downstream applications?
    2. DNA was not washed three times with the provided Wash Solution - Traces of salt from the binding step may remain in the sample if the column is not washed three times with the Wash Solution. Salt may interfere with downstream applications, and thus must be washed from the column.
    3. Ethanol was not added to the Wash Solution - Ensure that 28 mL of 95 - 100% ethanol is added to the supplied Wash Solution prior to use.
    4. Ethanol carryover - Ensure that the dry spin under the Column Wash procedure is performed, in order to remove traces of ethanol prior to elution. Ethanol is known to interfere with many downstream applications.
  4. How can I improve the DNA yield for my phages, which have a low plaque number?
    Concentrate the phages by centrifugation at 14,000 rpm for 10 minutes, then proceed with the protocol as written. Also, the amount of Proteinase K and the incubation time can be increased to improve the DNA yield.
  5. How can I eliminate host bacterial genomic DNA contamination from phage DNA?
    Treat enriched phage culture with DNaseI. Proceed with the regular phage DNA protocol.