Dahlquist:DNA Microarray Protocol
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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 Catalog #1615-5500)
- TipOne 101-1000 μL Filter Tips (sterile, free of detectable RNase, DNase, DNA & pyrogens; USA Scientific Catalog #1126-7810)
- TipOne 1-200 μL Graduated Filter Tips (sterile, free of detectable RNase, DNase, DNA & pyrogens; USA Scientific Catalog #1120-8810)
- TipOne 0.1-10 μL Filter Tips (sterile, free of detectable RNase, DNase, DNA & pyrogens; USA Scientific Catalog #1121-3810)
- 50 ml conical screw cap tubes, copolymer, bulk, sterile (USA Scientific Catalog #1500-1211)
- Powder-free latex exam gloves (USA Scientific Catalog #4900-2200, 4900-3300, 4900-4400)
- Kim wipes (Denville)
- aluminum foil
- compressed air duster
- 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)
- Nuclease-free water is provided in the Amino Allyl MessageAmp™ II aRNA Amplification Kit and is sufficient for the μL quantities called for throughout the protocol; additional water can be purchased from Applied Biosystems/Ambion, but it is expensive.
- DEPC is an oxidizer and should be fully removed from water used with DNA microarrays because the Cy5 dye is prone to degradation by oxidation.
- For larger quantities of nuclease-free water (non-DEPC treated) that are needed to dilute 20X SSC for washes, for example, I use Water, ASTM Type II, non-sterile, Reagent Grade, ACS (VWR Catalog #RC91505) that can be purchased in 10 L or 20 L cubes. This water was recommended by Genisphere in the DyeSaver 2 protocol as being validated for use with microarrays and not containing components that will oxidize Cy5; Kam D. Dahlquist 20:24, 15 August 2008 (EDT))
- 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
- Step 1
- Step 2
- Step 2 has some additional information that goes with it.
- Step 3
- Step 3 has multiple sub-steps within it.
- Enumerate each of those.
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- 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?
or instead, discuss this protocol.
