CRISPR× Fabio Liberante

Removing Endotoxins from Plasmid Preps

Want to boost transfection efficiency, nucleofection efficiency and even increase viral titre? Read on!

For those who aren't familiar with endotoxins, QIAGEN provide a good primer on the subject and why it matters. Long story short, endotoxin contamination in plasmid preps is one of the biggest factors in getting mammalian cells to play nice with foreign DNA. A number of years ago I came across this paper by Ma et al 2012 and it became part of my routine plasmid prep. I saw improvements in viral titre from my packaging cells, improved nucleofection rates and better transfection.

"the same endotoxin removal efficiency when compared with the temperature transition extraction method."

The beauty in their method is its simplicity. The solutions to be prepared are simple and cheap and keep for a long time.

Protocol

By example I have included the volumes needed for a 500µL plasmid maxiprep in brackets. Recipes are below.

  1. Add 0.2 volumes of TXS solution (100µL) and mix thoroughly by inverting
  2. Incubate at room temperature for 5~10 minutes. (I find overnight in the fridge works ok too)
  3. Add 0.2 volumes of 5M NaCl (120µL) and mix thoroughly by inverting
  4. Centrifuge mixture for 10 minutes at maximum speed
  5. Aspirate the clear upper layer, which contains the purified plasmid, into a clean tube
    Be careful not to aspirate any of the tinted TXS solution
  6. Add equal volume of isopropanol and mix by inverting
  7. Spin at full speed for 5 min
  8. Remove and discard supernatant
  9. Wash DNA pellet with 70% ethanol and spin for a minute
  10. Aspirate all residual 70% ethanol so that the pellet is almost dry
  11. Dissolve DNA pellet in a suitable amount of TE buffer or TRIS-only buffer (such as EB)

Recipes

Here are the recipes for the TXS solution and 5M NaCl.

Solution Volume Weight Reagent Supplier
TXS to 100mL H2O 3g [3% w/v] SDS Sigma-Aldrich (#74255)
until deep pink Oil Red O Sigma (#O0625)
6g [6% w/v] Triton X‑114 Sigma (#X114‑100ML)
5M NaCl to 100mL H2O 29.2g NaCl Sigma (#S3014‑500G)