Tom

PCR AlsT promotor with Pfu polymerase and primers 150, 250, 300, 500 bp from AlsT gene at 58 °C and 59 °C.

Result: PCR products were obtained for all primers except the 500 bp at both temperatures.

Arjan/Renske

Purified cDNA as before. cDNA concentrations can be read in the hybridization table (note: to be added Mo_16_july). The cDNA made today and the previously made cDNA were stained using the Cy3 and Cy5 dyes from Ambion and kept in the dark for 90 minutes.

Dyed cDNA was purified and concentration was measured (see table). Equal amounts of target/control where combined and the cDNA mixtures where dried in the speedvac rotor for 40 min, 60 degrees, 2000 rpm.

7 uL H2O and 35 uL Ambion hybridization buffer (37 degrees Celsius) where added to the dried cDNA mixture. The mixture was brought onto cleaned, pre-heated Bacillus subtilis slides. The slides used were provided by the Molgen research group. They were probed as described previously by Kloosterman et al., 2006 and produced as decribed Kuipers et al., 2002. The slides contained three technical replicates of the B.s. sp168 genome.

The cDNA was hybridized on the probed slides O/N at 48 degrees Celsius.

Kuipers, O., de Jong, A., Baerends, R., van Hijum, S., Zomer, A., Karsens, H., den Hengst, C., Kramer, N., Buist, G., & Kok, J. (2002). Transcriptome analysis and related databases of lactococcus lactis. Antonie Van Leeuwenhoek, 82(1-4), 113-122.

Kloosterman, T. G., Hendriksen, W. T., Bijlsma, J. J. E., Bootsma, H. J., van Hijum, S. A. F. T., Kok, J., Hermans, P. W. M., & Kuipers, O. P. (2006)." Regulation of glutamine and glutamate metabolism by GlnR and GlnA in streptococcus pneumoniae." Journal of Biological Chemistry, 281(35): 25097-25109.

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