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VersaTREK Detection IFU Manual / Product Insert

Induction of nitric oxide in human monocytes and monocyte cell lines by Mycobacterium tuberculosis. Citations & References

  • Authors: Jagannath C, Actor JK, Hunter RL
  • Journal: Nitric Oxide
  • PubMed ID: 9731635
Catalog #
  • G7921
  • N7927(Discontinued)
  • D7918(Discontinued)
  • N7892(Discontinued)

Clinical Significance of the Non-Tuberculous Mycobacteria and the Optimum Methods for Their Isolation [EN] Product Literature

Veterinary Microbiology Capabilities Product Literature

Bombyxin is a growth factor for wing imaginal disks in Lepidoptera. Citations & References

  • Authors: Nijhout H Frederik; Grunert Laura W;
  • Journal: Proc Natl Acad Sci U S A
  • PubMed ID: 12429853
Catalog #

How do you run a dose response curve for Geneticin (G418) or other selective antibiotic? Product FAQ

Answer

The amount of antibiotic required to be present in culture media to select for resistant cells varies with a number of factors, including cell type. Good laboratory practice requires that the optimal concentration of Selective Antibiotic required to maintain and select cells must be determined for each set of growth conditions. Whenever experimental conditions are altered (including use of Selective Antibiotic from a different lot), the optimal concentration of the product should be re-evaluated.

Below is a brief protocol for performing a kill curve with Geneticin. Follow the general protocol for other antibiotics as well but use appropriate ranges for each antibiotic. For example, Geneticin: 100-1,500 ug/ml, Blasticidin: 1-10ug, Zeocin resistance gene: 100-1000 ug/ml.

Kill Curve Assay:

1. Dissolve Geneticin Selective Antibiotic in fully supplemented growth medium without antibiotics at a concentration of 5 mg/ml and filter using a 0.22 micron filter.
2. Prepare 6-well cell culture plates by adding Geneticin Selective Antibiotic to the growth medium to desired concentrations. A range from 100-1,200 µg/ml in 100 µg increments is recommended.
3. Treat cells with trypsin and dilute to a concentration of 4000 cells/ml.
4. Add 100 µl of cell suspension to each well and incubate plates in a humidified CO2 atmosphere at 37°C.
5. At 10 to 14 days, aspirate the supernatant and wash the cells with phosphate buffered saline and stain the cells with 0.5% methylene blue and 50% methanol for 20 minutes.
6. Score the plates by calculating percentage of survival by number of individual colonies for percent confluence.
7. Calculate the percentage of survival in the presence of each dilution of Geneticin Selective Antibiotic versus the percentage of survival in the absence of Geneticin.
8. Generate a dose response curve by plotting the percentage of survival on the y axis versus the concentration of Geneticin Selective Antibiotic in µg/ml on the x axis for both the sensitive and resistant cell lines.

If you are performing sequential transfections, one needs to establish dose response curve for the first antibiotic, create a stable and then perform a second does response curve on that stable in the presence of 2 antibiotics.

Answer Id:: E4300

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Brochure: para-JEM Brochure Product Literature

Brochure: Mycobacteria Testing Workflow Solutions Product Literature

Is CTS AIM-V Medium, without phenol red, without antibiotics ready-to-use? Product FAQ

Answer

Yes, CTS AIM-V Medium, without phenol red, without antibiotics is ready-to-use. Additional supplementation with cytokines and growth factors may be required for certain types of cells. Human serum or CTS Immune Cell SR (Cat. Nos. A2596101 and A2596102) can be added to AIM-V medium to promote cell expansion.

Answer Id:: E16474

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VersaTREK REDOX and Myco Media Specifications Product Literature

VersaTREK Automated Microbial Detection System Product Literature

Apoptosis induces efflux of the mitochondrial matrix enzyme deoxyguanosine kinase. Citations & References

  • Authors: Jüllig M, Eriksson S,
  • Journal: J Biol Chem
  • PubMed ID: 11294860
Catalog #

I performed an LR reaction, followed by transfection into Sf9/Sf21 insect cells, but do not see any signs of infection even though it's been 72 hours. What should i do? Product FAQ

Answer

Please see our recommendations below:

- Check the LR reaction by PCR analysis prior to transfection into insect cells.
- We recommend using Grace's Insect Cell Culture Medium, Unsupplemented during the transfection experiment instead of serum-free medium, as components in serum-free medium may interfere with transfection.
- Ensure that FBS, supplements, or antibiotics are not included during transfection, as the proteins in these materials can interfere with the Cellfectin II Reagent.
- Use the LR recombination reaction using the pENTR/CAT plasmid as a positive control and Cellfectin II Reagent only (mock transfection) as a negative control.
- Ensure that cells are in the log phase of growth with >95% viability, and the amount of cells are in accordance with the suggestions in the manual.
- Cells may not show signs of viral infection for up to a week depending on transfection efficiency; continue culturing and monitor cells daily for signs of infection.

Answer Id:: E9459

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I'm getting a low yield of virus when using the Bac-to-Bac baculovirus expression system. Why is this, and what can I do to improve my yield? Product FAQ

Answer

Please see the following reasons and suggestions to improve yield:

- Low transfection efficiency: We recommend using our Cellfectin II Reagent for transfection; perform the transfection in Grace's Medium, Unsupplemented and ensure no supplements, FBS, or antibiotics are present during the transfection. Harvest the viral supernatant when signs of infection are visible (typically >96 hr post-transfection).
- Cells are plated too sparsely; check recommended cell densities.
- Use too much or too little Cellfectin II Transfection Reagent: Optimize amount used.
- Time of incubation with DNA:lipid complexes is too short or too long: Optimize incubation time (3-8 hr).
- Recombinant bacmid DNA is degraded: Check the quality of your recombinant DNA by agarose gel electrophoresis prior to transfection; prepare bacmid DNA using PureLink HiPure Plasmid DNA Miniprep or Maxiprep Kit.
- Bacmid DNA is not pure (contains recombinant and empty bacmid): Screen other DH10Bac transformants and perform plaque purification to isolate recombinant baculovirus.

Answer Id:: E9451

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Brochure: Blood Culture Solutions Product Literature

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