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How should I resuspend my siRNA library? Product FAQ

Answer

RNA oligonucleotides are susceptible to degradation by exogenous ribonucleases introduced during handling. Wear gloves when handling this product. Use RNase-free reagents, tubes, and barrier pipette tips. Upon receipt, your siRNAs may be safely stored in a non-frost-free freezer at or below -20 degrees C (dried oligonucleotides are shipped at ambient temperature). Invitrogen siRNA reagents are shipped as dry pellets at ambient temperature and should be stored at -20 degrees C upon arrival in a manual defrost or noncycling freezer. Under these conditions, the siRNAs are stable for at least 3 years. If necessary siRNAs as dry pellets (unopened) can be stored at 4 degrees C for at least a year.

To resuspend Invitrogen siRNAs provided in plates:
Centrifuge each plate at low speed (maximum RCF 4,000 x g) to collect the contents at the bottom of the wells before removing the seal.
Wipe the adhesive foil cover with 70% ethanol or other RNase-decontamination solution such as RNaseZap RNase Decontamination Solution (Cat. No. AM9780, AM9782, AM9784).
1. Thermo Scientific or carefully peel back the foil seal to gain access to wells. Use caution and avoid shredding the seal.
2. Add nuclease-free, sterile water, using a multichannel pipettor or liquid handling system and sterile tips, to achieve the desired concentration. Resuspend siRNAs to a convenient stock concentration using the recommended volume of Invitrogen Nuclease-Free Water (not DEPC-treated). Concentrated stocks of 10 µM or more are recommended. However, stock solutions of 2-5 µM may better accommodate dilution schemes for high?throughput transfections and assays conducted on robotic platforms.
3. Gently pipet up and down 5 times to resuspend. Place the solution on an orbital mixer/shaker for 70-90 minutes at room temperature. This additional mixing results in more reliable resuspension.
4. Centrifuge briefly to collect the liquid at the bottom of the wells, if necessary.
5. (Optional) Aliquot the siRNAs into one or more daughter plates, to limit the number of freeze/thaw cycles to which the siRNAs are subjected.
6. Store at -20 degrees C or lower for long-term storage. The siRNAs can be stored at 4 degrees C for short-term use, but care should be taken to seal well to avoid evaporation.

Answer Id:: E10043

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Why would the Luminex acquisition software display "Sample Empty" messages during analysis? Product FAQ

Answer

(1) The user did not properly aliquot the diluted beads, such that no beads were actually added to the wells (make sure that the bead concentrates are sonicated and vortexed well, then check the pipet tip to ensure that air bubbles were not drawn up)

(2) The user missed loading diluted beads to some wells, which is likely since the small volume is clear and difficult to visualize in the clear plastic plate (we have now addressed this customer difficulty by coloring each of the Buffer Reagent Kit components)

(3) The user applied too much vacuum pressure at some point during the wash steps, or allowed the pressure to spike even once, such that the filter membrane tore in a few wells releasing the beads (make sure that the vacuum manifold pressure is kept below 5mm/in Hg, depending on their system -- a good rule of thumb is that it should take a full 3-second count to GENTLY empty the wells of 200uL)

(4) The user did not properly sonicate and vortex the beads prior to dilution, such that the percent of bead aggregation was high and the instrument was unable to find enough single beads to meet the events/bead value designated by the customer (make sure that the Bead Concentrate tube is put into the waterbath all the way to the cap, since the tube is hollow until the top third)

(5) The user lost beads by shaking the plate too aggressively or handling it improperly (make sure that the orbital shaker is set to a speed that allows for maximum vortex in the wells without spillage)

(6) The user exposed the beads to an excess of light during storage or running of the assay, such that some but not all of the beads were photobleached and therefore falling outside the acceptable range for each bead region (make sure that the plate is covered on the top/sides with foil throughout the assay, away from Windows and spotlights, and that the bead component of the kits is stored in the dark)*

(7) There was a clog in the sample needle, such that the instrument was unable to take up enough sample to meet the number of events requested per bead region (suggest that the user follow the manual instructions for dislodging a clog, which include several Back Flush steps and may require removal of the needle for sonication with probe alignment).

* Some of the older Antibody Bead Kits still have clear plastic tops instead of black ones. In cases where customers store kits in lit refrigerators, or keep them open on the lab bench, even a few hours of light exposure is enough to photobleach beads. It is important to note, in general, that higher number bead regions are more susceptible to photobleaching. In order to draw conclusions about the source of the difficulty, we would ask to see the data, specifically the Masterplex QT file, which would enable us to examine the pattern of "Sample Empty" occurrences in addition to the bead counts per well.

Answer Id:: E5158

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Catalog: Remel, Oxoid, VersaTREK and Sensititre Microbiology Product Catalog, US Product Literature

A novel cell-associated protection assay demonstrates the ability of certain antibiotics to protect ocular surface cell lines from subsequent clinical Staphylococcus aureus challenge. Citations & References

  • Authors: Wingard JB, Romanowski EG, Kowalski RP, Mah FS, Ling Y, Bilonick RA, Shanks RM
  • Journal: Antimicrob Agents Chemother
  • PubMed ID: 21628536

Evaluation of Dipicolinic Acid-Based Mueller Hinton Agar Biplate for Detection of IMP-1 and VIM-2 type Metallo-β-Lactamase in Imipenem Non-susceptible Gram Negative Bacilli Citations & References

  • Authors: Shin, KS; Son, BR; Koo, SH; Lee, SH; Ahn, JB; Park, SH; Hwang, SY
  • Journal: Korean Journal of Laboratory Medicine
Catalog #
  • 4339386
  • 4413750
  • N8050400(Discontinued)
  • N8050200(Discontinued)
  • N8050002(Discontinued)
  • ZGEXSC9700384W
  • ZGEXSC9700384W3Y
  • N8050002R
  • 4441166

Biological and molecular characteristics of Mycobacterium tuberculosis clinical isolates with low-level resistance to isoniazid in Japan Citations & References

  • Authors: Abe, C; Kobayashi, I; Mitarai, S; Wada, M; Kawabe, Y; Takashima, T; Suzuki, K; Sng, LH; Wang, S; Htay, HH; Ogata, H
  • Journal: JOURNAL OF CLINICAL MICROBIOLOGY

The structure of the culturable root bacterial endophyte community of Nicotiana attenuata is organized by soil composition and host plant ethylene production and perception Citations & References

  • Authors: Long, HH; Sonntag, DG; Schmidt, DD; Baldwin, IT
  • Journal: New Phytologist

Human methicillin-sensitive Staphylococcus aureus biofilms: potential associations with antibiotic resistance persistence and surface polysaccharide antigens. Citations & References

  • Authors: Babra C, Tiwari J, Costantino P, Sunagar R, Isloor S, Hegde N, Mukkur T
  • Journal: J Basic Microbiol
  • PubMed ID: 23686411

Glutathione levels and BAX activation during apoptosis due to oxidative stress in cells expressing wild-type and mutant cystic fibrosis transmembrane conductance regulator. Citations & References

  • Authors: Jungas Thomas; Motta Iris; Duffieux Francis; Fanen Pascale; Stoven Véronique; Ojcius David M;
  • Journal: J Biol Chem
  • PubMed ID: 12023951
Catalog # 11514015

Coagulase-Negative Staphylococci Isolated From Ocular Wound Infections After Laser Refractive Surgery: Attachment to and Accumulation on Soft Contact Lenses Citations & References

  • Authors: Faghri, J; Razavi, MR
  • Journal: Eye & Contact Lens-science and Clinical Practice

Silencing Toll-like receptor-9 in Pseudomonas aeruginosa keratitis. Citations & References

  • Authors: Huang X, Barrett RP, McClellan SA, Hazlett LD
  • Journal: Invest Ophthalmol Vis Sci
  • PubMed ID: 16249500

Click Chemistry—Section 3.1 Molecular Probes Handbook

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