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Arg-PNA Microarrays
hybridisation experiments were carried out in 10mM phosphate buffer, 100mM NaCl, 0.1mM
EDTA, pH= 7. All the hybrid samples reported were first incubated at 90°C for 5 minutes,
then slowly cooled to room temperature. The samples were heated (1°C/min) and the UV
signal variation at 260nm was recorded. Melting temperatures were measured as the
maximum of the first derivatives of the melting curves.
4.4.4 PNA spotting on microarray slides
Each PNA was dissolved in a solution containing carbonate buffer (0.1M, pH= 9), SDS
0.001% at a concentration of 50 µM and spotted by using a pin system spotter SpoArray 24
(Perkin-Elmer, Waltham, USA) onto commercial slides (CodeLink, Amersham Bioscences,
New Jersey, USA). Then the following washing steps were performed: washing solution 1:
carbonate 0.1M and SDS 0.2%; washing solution 2: carbonate 0.1M and SDS 1%; washing
solution 3: carbonate 0.1M and SDS 0.001%. Fixing was performed overnight at a controlled
moisture ( ∼ 75%). Capping of the unreacted sites was done by using a solution of Tris 0.1M,
ethanolamine 50mM, pH=9, at 50°C for 1h. Conditioning of the slides was performed at 50°C
with SSC buffer (0.6M NaCl, 0.06M sodium citrate, pH= 7) and SDS 1% solution for 1h.
Final washings were carried out with doubly distilled water for 5 minutes.
4.4.5 Oligonucleotide hybridization on microarray slides
Each Cy5 labeled oligonucletide was dissolved in a solution containing SSC buffer (0.3M
NaCl, 0.03M sodium citrate, pH= 7), SDS 0.1% at a concentration of 100nM. Before
hybridization the slides were hydrated with a solution of SSC buffer (0.3M NaCl, 0.03M
sodium citrate, pH= 7), SDS 0.1% at 40°C for 30 minutes. Then, oligonucleotide solutions
were deposited on the slides by using a hybridization chamber and left at 50°c for 2 hours.
After hybridization, washing steps were performed at 50°C for 5 minutes with a solution of
SSC buffer (0.3M NaCl, 0.03M sodium citrate, pH= 7), SDS 0.1%. Final washing was carried
out for 1 minute at room temperature with a two different SSC buffer solutions: 0.03M NaCl,
0.003M sodium citrate, pH= 7 and 0.01M NaCl, 0.001M sodium citrate, pH= 7.
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