Dahlquist:DNA Microarray Protocol

Overview

This protocol describes all the steps necessary to perform aRNA synthesis, labeling, hybridization, and scanning of DNA microarrays for Saccharomyces cerevisiae, beginning with total RNA.

NOTE: This protocol is still under construction!

Materials

Supplies & Reagents

• SealRite 1.5 mL Natural Microcentrifuge Tubes (free of detectable RNase, DNase, DNA & pyrogens; USA Scientific #1615-5500)
• TipOne 101-1000 μL Filter Tips (sterile, free of detectable RNase, DNase, DNA & pyrogens; USA Scientific #1126-7810)
• TipOne 1-200 μL Graduated Filter Tips (sterile, free of detectable RNase, DNase, DNA & pyrogens; USA Scientific #1120-8810)
• TipOne 0.1-10 μL Filter Tips (sterile, free of detectable RNase, DNase, DNA & pyrogens; USA Scientific #1121-3810)
• Amino Allyl MessageAmp™ II aRNA Amplification Kit (Applied Biosystems Catalog #AM1753)
  • 20 reactions per kit
  • Applied Biosystems Catalog Web Page. Originally an Ambion product. Ambion was acquired by Applied Biosystems and the kit can only be purchased through them.
  • Ambion Product Web Page. Contains useful links.
  • Ambion Protocol (PDF). Protocol packaged with kit.
• CyDye Post-Labeling Reactive Dye Pack (GE Healthcare Life Sciences Catalog #RPN5661)
  • 12 X Cy3 + 12 X Cy5 per box
  • GE Healthcare Product Web Page. Distributed through VWR.
  • Use protocol for Amino Allyl MessageAmp™ II aRNA Amplification Kit.
• TE Buffer
• Microcuvettes
• Nuclease-free water (non-DEPC treated)
• Fragmentation Reagents
• DIG Easy Hyb
• Sonicated salmon sperm DNA
• oligo dA

Equipment

• Dedicated set of RNase-free pipetmen
• Speedvac
• Water bath set to 50°C
• Water bath set to 37°C
• Heat block set to 70°C

Procedure

1. Step 1
2. Step 2
   • Step 2 has some additional information that goes with it.
3. Step 3
   1. Step 3 has multiple sub-steps within it.
   2. Enumerate each of those.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

1. List troubleshooting tips here.
2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers and books

If this protocol has papers or books associated with it, list those references here. See the OpenWetWare:Biblio page for more information.

Contact

• Who has experience with this protocol?
  • Kam D. Dahlquist

or instead, discuss this protocol.