- What is Plasmid+®?
- Plasmid+® liquid media is an enriched media specifically designed for plasmid DNA production. Plasmid+® supports much higher cell densities and plasmid yields than LB media. Optimal shake flask yields are achieved using Ultra Yield™ Flasks (Thomson Instrument Company), which facilitates maximum culture aeration. Plasmid+® media may also be used in a bioreactor with continuous aeration and agitation.
- How do I store Plasmid+®?
- Store Plasmid+® liquid media at room temperature for up to 12 months after manufacture date.
- What bacterial strains can be used with Plasmid+®?
- E. coli DH5α is the preferred host strain for use with Plasmid+® media. E. coli XL1-Blue also produces high quality plasmid DNA and may improve plasmid DNA yields with plasmids smaller than 3kb. Other strains can be used, but for DNA production the ones above are the best available.
- Do I need to use a seed culture to inoculate?
- A seed culture is recommended for culture volumes larger than 50mL. Cultures less than 50mL may be inoculated directly from a glycerol stock or plate. To prepare a seed culture, use a glycerol stock or plate to inoculate 1/100th of the final culture volume of LB + appropriate antibiotic (e.g. 100 μg/mL ampicillin; 50 μg/mL kanamycin) and grow to saturation with shaking at 37°C.
- How do I use Plasmid+® with the Ultra Yield Flasks?
- Using aseptic technique, add Plasmid+® media and appropriate antibiotic (e.g. 100μg/mL ampicillin; 50μg/mL kanamycin) to one or more Ultra Yield™ Flasks; see Table 1 for recommended culture volumes. Inoculate the media, place an AirOtop seal on the flask, and grow at 37°C with shaking at 350 rpm for 16-18 hours.
- Do I need to adjust my purification protocol? YES!
- Plasmid DNA may be purified from Plasmid+® cultures by the common methods (e.g. Qiagen® Mega kits or Qiagen® Giga kits, etc.). Mega kits we have found starting with 5mgs works well at the high range. Giga kits we have found starting with 20mgs works well at the high range. However, because Plasmid+® media typically yields 5-10 times increased yields when compared to LB media, the increased cell mass and plasmid DNA content must be taken into consideration to insure efficient lysis and to avoid overloading purification columns. When using plasmid purification kits, the culture volume per purification should be decreased by a factor of 5 with respect to the recommended LB culture volume.
- How do I avoid overloading the purification columns, so I don’t lose my DNA?
- Plasmid+® creates an enriched DNA cell paste, it is recommended that you resuspend the cell pellet using 10 mL of P1 buffer per gram of cell pellet. If preferred, using a volume of P1 buffer equivalent to half of the Plasmid+® culture volume is acceptable. See diagram on Improved DNA Protocol for E.coli with Plasmid+® Media.
- Check that the proper antibiotic and concentration is used
- Insure proper culture aeration.
- Use the recommended media volumes in Ultra Yield™ Flasks with shaking at 350 rpm
- Increase the growth time (for up to 48 hours)
- Use a starter culture for final culture volumes > 50 mL
- Protein may be toxic. Try growth at 16 °C. Growth time may need to be increased at 16 °C
- Make sure resuspension of cell pellet is complete
- Use enough resin for higher quantity yields
Low Protein Yield
Low Recovery From Purification
Thomson is not affiliated with Qiagen® or their products or manufacturers of LB broth.