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Protocol
Dual tag microarray
Keywords: RCA, Padlock probe, Microarray
The Dual tag microarray readout is used for high precision readout of reporter nucleic acids comprising two tag motifs, in the 3’ and 5’end respectively.
Instructions
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Microarray ligation (5Y8R71)
On-chip ligation is performed by adding 30 µl ligation mix, comprising Ampligase, buffer (Epicentre Biotechnologies, WI, USA), 25 µg BSA, and 5 U Ampligase to 20µl of ligation substrates and incubating at 50°C over night after addition to the microarray.
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Microarray wash (5Y93TV)
Each microarray is gently flushed with 1 ml of 0.75x TNT buffer at RT (110 mM NaCl, 7.5 mM Tris HCl pH 8, 0.04% Tween 20). Microarrays are then dried by centrifugation using a tabletop centrifuge.
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On-chip RCA (5Y95XX)
Circularized DNA is replicated by on-chip RCA in 50 µl phi29 buffer (New England Biolabs, MA, USA), 0.5 µg/µl BSA, 0.2 mM of each dNTP and 10 U of phi29 polymerase for 45 min in 37 ºC.
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Microarray wash (5Y93TV)
Each microarray is gently flushed with 1 ml of 0.75x TNT buffer at RT (110 mM NaCl, 7.5 mM Tris HCl pH 8, 0.04% Tween 20). Microarrays are then dried by centrifugation using a tabletop centrifuge.
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Detection probe hybridization (5Y9ABP)
Amplification are visualized by hybridizing 10 nM of a complementary detection probe labeled with Cy5 in 2x SSC (0.3 M NaCl, 30 mM Na-citrate) and 0.1% SDS for at least 1 h at 50°C.
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Microarray wash (5Y93TV)
Each microarray is gently flushed with 1 ml of 0.75x TNT buffer at RT (110 mM NaCl, 7.5 mM Tris HCl pH 8, 0.04% Tween 20). Microarrays are then dried by centrifugation using a tabletop centrifuge.
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Image acquisition (5Y9DCT)
Microarrays are scanned with a scanner ( e.g. Genepix scanner, Molecular Devices Corporation, CA, U.S.A.) at 5 µm resolution and analyzed with the appropriate software, (e.g. Genepix Pro 6.0).
