ProceduresEColiMicroarray < Lab

---++ Materials

| *Description* | *Cat #* | *Price* |
| Qiagen RNeasy Protect Bacteria Mini Kit (50) | 74524 | $386.00 |
| Invitrogen Superscript Plus Indirect cDNA Labeling System (30) | | |

The Qiagen kit contains enough RNA protect reagent for processing 100 ml of culture.

---++ RNA Isolation

   1 Revive and precondition cultures to be tested.
   1 Add 100 µl of overnight culture to 10 ml of fresh media (DM100) in 50 ml flask.
   1 Growth for approximately 5-6 hrs for REL606/607 or evolved strains or 6-7 hrs for Rif<sup>R</sup> ancestors with large fitness defects. 
   1 <u>Quickly</u> take a small aliquot from each sample for measuring in the spectrophotometer and return cultures to incubator. Measure OD<sub>600</sub>. When harvesting, the OD<sub>600</sub> should be approximately 0.5-0.6 to get exponential phase RNA. The final OD in this media after 24 hrs of growth reaches approximately 0.16.
   1 Take 8 ml of remaining culture. This will contain approximately 5 x 10<sup>8</sup> cells. Divide equally into two 15 ml conical tubes. Add 2x volume of RNA protect reagent to each. 
   1 Follow remaining steps of RNeasy Mini kit protocol for processing 5 x 10<sup>8</sup> cells. 

---++ RNA Quality Check

At the CAFG, use the Agilent 2100 Bioanalyzer to check the quality of RNA samples. There are twelve samples per $30 chip. Select the "Bacterial RNA assay option". The ratio of the 16S RNA to 23S RNA peaks should be 1.9 in a high quality sample.

---++ cDNA Synthesis

   * Use only random hexamers.
   * Save 1/10 reaction before every purification step.

---++ RNA Labeling

---++ RNA Hybridization

---++ Slide Scanning
Barrick Lab > ProtocolList > ProceduresEColiMicroarray
Topic revision: r2 - 2009-07-09 - 21:45:28 - Main.JeffreyBarrick