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5 Alteration of program parameters Effect Increase hybridization temperature Can prevent false positive signals, decrease background, can also increase dynamic range Decrease hybridization temperature Increase overall intensity, but background can increase too Increase wash temperature Decrease background, but can also decrease signal Increase washing duration Decrease background, but can also decrease signal Decrease wash temperature Enhance spot signal, can lower dynamic range, can increase background Enhance spot signal, can lower dynamic range, can Decrease washing duration increase background, but can prevent damage to the slide surface Increase detergent concentration of 1st wash buffer, if used slide surface is very hydrophobic Reduces air bubbles in the chamber, because of reduction of repulsion of aqueous solutions Increase probe injection temperature Prevention of signal gradient formation Chamber conditioning in water bath at 70 C (o/n) Prevention of air bubble formation during long hybridizations at high temperature Increasing concentration of secondary labeling molecules Can increase signal, can prevent signal gradient formation 1 5

6 Manual procedure Pre-hybridization of microarray glass slide: Pre-hybridize microarray glass slide 1.5hrs at 45 C. Rinse pre-hybridized microarray glass slide in dh2o Rinse microarray glass slide in 70% ethanol Air dry glass slide Lifterslips: Store lifterslips in soap solution. Rinse lifterslips Air dry lifterslips Automation Hybridization microarray glass slide: Preheat hybridization buffer Heat probe mix 2min 95 C Apply this probe mix to microarray glass slide Add H2O to each well in the hybridization chamber Hybridize overnight 45 C in water bath Wash steps after hybridization microarray glass slides: Remove lifterslip gently from microarray glass slide in 2xSSC/0.1% SDS 45 C Transfer microarray glass slides to 1xSSC and wash twice for 10min 45 C Subsequently, transfer glass slides to 0.2xSSC and wash twice for 2:45min Finally, dip glass slides in 0.1xSSC at RT Spin dry glass slides 3 800rpm in 50ml tube Manual / Automated Manual / Automated Manual 2 6

7 Process step Manual method Tecan Hybridization Station Pre-hybridization 1 st wash with pre-hybridization buffer (4xSSC / 0,2%SDS) 20 seconds at 65 C Probe-injection 20 µl probe under cover slip Probe injection at 65 C (same DNA amount as in manual method) followed by 2 minutes Hybridization Hybridization for 16 hours at 65 C in water bath Wash All at room temperature in TeleChem WashStation denaturation Hybridization 16 hours at 65 C with agitation mode medium 2 x 5 minutes with 2xSSC / 0,1%SDS 3 x Wash 2 minutes (2xSSC / 0,1%SDS) at 33 C 2 x 1 minute with 0,2xSSC 2 x Wash 2 minutes (0,2xSSC) at 33 C 1 x 1 minute with 0,1xSSC / 0,1% Tween 2 x Wash 2 minutes (0,1xSSC / 0,1% Tween) at 33 C 1 x 5 minutes with 0,1xSSC 1 x Wash 1:30 minutes (0,1xSSC) at 33 C Soak 0 minute Drying Drying in centrifuge Drying at 30 C for 1:30 minutes with N 2 7

8 Process step Manual method Tecan Hybridization Station Pre-hybridization Probe 1 minute at 95 C (heating block) 1 st wash with pre-hybridization buffer (SSC / SDS Solution without DNA and blocking solution) 20 seconds at 60 C Probe-injection ~ 700 µl probe in seal frame Probe injection (same DNA amount as in manual method); then denaturation for 2 minutes at 95 C. Hybridization Hybridization over night at 60 C Hybridization for 14 hours at 55 C with agitation mode medium Wash 1 x 5 minutes at 65 C with buffer 1 (2xSSC/0,5%SDS) 2 x wash with buffer 1 at 60 C Wash 1 minute s 1 x 5 minutes at room temperature with buffer 2 (0,5xSSC) 1 x 5 minutes at RT with buffer 3 (0,1xSSC) Immerse in H 2 O very shortly 2 x wash with buffer 2 at 25 C Wash 1 minute s 1 x wash with buffer 3 at 25 C Wash 1 minute Soak 2 minutes Wash with H 2 O for ~ 10 seconds Drying Drying in centrifuge Drying at 30 C for 1:30 minutes with N 2 8

9 Process step Manual method Tecan Hybridization Station 1 st hybridization Pre-hybridization Probe 10 minutes at 65 C (heating block) 1 st wash with pre-hybridization buffer (SSC / SDS Solution without formamide) 20 seconds at 55 C Probe-injection 30 µl probe under cover slip Probe injection (same DNA amount as in manual method) Hybridization Hybridization for 90 minutes at 55 C Hybridization for 90 minutes at 55 C with agitation mode high Wash Immerse in H 2 O very shortly Wash 2 x 5 minutes at room temperature with buffer 1 Immerse in H 2 O very shortly 2 x wash with buffer 1 at RT.: Wash 1 minute Soak 2 minutes 2 nd Labeling Wash Drying in centrifuge 2 nd staining solution for 30 minutes at RT under cover slip (Streptavidin Cy5) Wash 2 x 5 minutes at room temperature with buffer 2 Immerse in H 2 O very shortly Wash 30 seconds with buffer like staining solution without Streptavidin Cy5 Probe injection (streptavidin-cy5-solution) Hybridization for 30 minutes at RT with agitation mode high 2 x wash with buffer 2 at room temperature: Wash 1 minute Soak 2 minutes Wash with H 2 O for ~ 10 seconds. Drying Drying in centrifuge Drying at 30 C for 1:30 minutes with N 2 9

10 Process step Manual method Tecan Hybridization Station Pre-hybridization 1 st wash with pre-hybridization buffer (5xSSC) 20 seconds at 42 C Probe injection 30 µl probe under cover slip Injection of probe solution at 42 C (same Hybridization Wash Hybridization for 20 hours or over night at 42 C in shaking water bath All at 30 C in shaking water bath DNA amount as in manual method) Hybridization for 2 and 14 hours at 42 C with agitation. Agitation mode high for 2h hybridization Agitation mode medium for 14h hybridization 1 x 5 minutes with 2xSSC / 0,2%SDS 2 x Wash 1 minute (2xSSC / 0,2%SDS) at 30 C 1 x 5 minutes with 1xSSC 2 x Flow 1 minute (1xSSC) at 30 C 1 x 5 minutes with 0,5xSSC 2 x Flow 1 minute (0,5xSSC) at 30 C Drying Drying in centrifuge Drying at 30 C for 1:30 minutes with N 2 10

11 Technical note Austria T Belgium T China T Denmark France Germany Italy Japan Netherlands Portugal Singapore Spain Sweden Switzerland UK USA ROW

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