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One of the most common issues we deal with in the lab environment on a daily basis is sample DNA concentration.  To ensure that we have sufficient amounts of DNA, we employ a simple DNA Concentration Protocol.

In essentially all cases our laboratory protocols and vendor kits specify minimum DNA concentration requirements.  Sample DNA concentrations must meet or exceed the minimum requirements in order to proceed with various protocols.  This is applicable to DNA extraction, DNA clean-up, PCR inhibitor removal, DNA quantitation, whole genome sequencing, targeted resequencing including gene panels, HLA typing and myriad other functions in the lab environment.

We routinely receive extracted gDNA samples from researchers and clients.  In some cases the sample DNA concentrations fall below the minimum requirements for a particular lab protocol.  This is especially common when researchers use spectrophotometric methods for DNA quantitation, whereas we generally use fluorometric methods for quantitation;  the disparities in measured DNA concentration between these two methods can be significant. We then use the DNA Concentration Protocol to increase DNA concentration above the minimums.

We also routinely perform DNA extraction protocols on a wide variety of sample types, including cells, bodily fluids, bacterial samples, fungal samples, etc.  Occasionally the extracted gDNA concentration falls below the minimum protocol requirements.  This is especially true if the sample cell count is too low.  In these cases we again use the DNA Concentration Protocol to get the extracted DNA concentration above minimums.

 

Protocol

For those who are interested, here is a general outline of the protocol:

  1. Add 1/10 volume of 3 M sodium acetate (pH = 5.2) to the sample in solution. 
  2. Add 1 uL of glycogen solution (20 mg/mL) per 20 μL of sample in solution. 
  3. Add 2.5 volumes of ethanol. 
  4. Incubate at -20° C for 1 hour.
  5. Centrifuge 15 minutes at 10,000 rpm.
  6. Discard the supernatant without disturbing the pellet. 
  7. Rinse the pellet with cold 70% ethanol. 
  8. Centrifuge 5 minutes at 10,000 rpm.
  9. Discard the supernatant without disturbing the pellet. 
  10. Air dry the pellet for 3 minutes, being careful to not over-dry the pellet. 
  11. Dissolve the pellet in 35 uL TE buffer. 

We find that the addition of glycogen solution significantly improves the yield of highly concentrated DNA.

This is a relatively simple, quick and cost effective method for concentrating DNA.  In almost all cases it yields sufficient DNA concentrations to continue with our lab procedures.