Compatibility of DMSO spotting buffers with Nexterion Slide E Epoxy-coated Microarray slides
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Compatibility of DMSO spotting buffers with SCHOTT Nexterion Epoxy-silane coated microarray slides. Effect of using different spotting pins and DMSO concentrations.
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Compatibility of DMSO spotting buffers with Nexterion Slide E Epoxy-coated Microarray slides
- 1. November 2004DMSO Spotting BufferMicroarray Solution SCHOTT Jenaer Glas GmbH Final Report DMSO Spotting Buffers on Slide E Norman Rahnis, Kornelia Kuhn, Katrin Steinmetzer Application Support/R&D Microarray Solutions Schott Jenaer Glas GmbH
- 2. November 2004DMSO Spotting BufferMicroarray Solution SCHOTT Jenaer Glas GmbH Spotting Solution Components DMSO, SSC, phosphate buffer, SDS Slides Nexterion Slide E Probes 70mer (from E. coli Genome Oligo Set Version 1.0) 10 µM Spotting QArray Mini, 22°C, 45% RH, SMP3 pin (Telechem), aQu Split Pin K2801 (diameter 150 µm) Target total RNA (E.coli) was used to synthesize aa-cDNA, the coupling of NHS-ester-dyes (Nexterion Dye3 and Dye5) was performed with a standard protocol derived from a Cy3/Cy5 labeling protocol, Hybridization Tecan Hybridization Station HS4800, in 5xSSC, 0.1% SDS, 0.1 mg/ml herring sperm DNA, 50% formamide, 42°C, 16 hrs Scanning Affymetrix Array Scanner 428 Analysis IconoClust Experimental Details
- 3. November 2004DMSO Spotting BufferMicroarray Solution SCHOTT Jenaer Glas GmbH ....some remarks the following images show results after hybridization blue spots mean lower signal, green spots mean higher signals suitable buffers were highlighted in red spot diameter was only determined for good spots
- 4. November 2004DMSO Spotting BufferMicroarray Solution SCHOTT Jenaer Glas GmbH Results SMP3 Pin 50% DMSO 50% DMSO 3x SSC 50% DMSO 1.5x SSC, spot diameter 70 µm 50% DMSO 150 mM PP, 0.1% SDS 50% DMSO 150 mM PP, 0.05% SDS 50% DMSO 150 mM PP 50% DMSO
- 5. November 2004DMSO Spotting BufferMicroarray Solution SCHOTT Jenaer Glas GmbH Results SMP3 Pin 30% DMSO 30% DMSO 3x SSC 30% DMSO 1.5x SSC, spot diameter 70 µm 30% DMSO 150 mM PP, 0.1% SDS 30% DMSO 150 mM PP, 0.05% SDS 30% DMSO 150 mM PP 30% DMSO
- 6. November 2004DMSO Spotting BufferMicroarray Solution SCHOTT Jenaer Glas GmbH Results SMP3 Pin 25% DMSO 0.005% SDS 50% DMSO 1.5x SSC, 0.005% SDS, spot diameter 60-70 µm 25% DMSO 1.5xSSC, 0.005% SDS Spot diameter 90 µm 50% DMSO 75 mM PP best results were obtained when combining DMSO with SSC and with or without SDS (but only for f.c. SDS of 0.005%, higher concentrations lead to precipitation) a combination of DMSO, phosphate buffer and SDS does not work further tests with additional combinations of DMSO, SSC and other detergents will be tested
- 7. November 2004DMSO Spotting BufferMicroarray Solution SCHOTT Jenaer Glas GmbH Results SMP3 Pin 50% DMSO 2x SSC best results were obtained when combining DMSO with SSC and with or without detergents spot sizes range between 100 and 140 µm similar results were obtained with 30% DMSO 50% DMSO 1.5x SSC, 0.025% CTAB 50% DMSO 1.5x SSC, 0.025% Triton X100 50% DMSO 2.5x SSC 50% DMSO 1.5x SSC, 0.005% SDS
- 8. November 2004DMSO Spotting BufferMicroarray Solution SCHOTT Jenaer Glas GmbH Results aQu K2801 Pin 50% DMSO 50% DMSO 3x SSC 50% DMSO 1.5x SSC 50% DMSO 150 mM PP, 0.1% SDS 50% DMSO 150 mM PP, 0.05% SDS 50% DMSO 150 mM PP 50% DMSO no suitable buffer combinations
- 9. November 2004DMSO Spotting BufferMicroarray Solution SCHOTT Jenaer Glas GmbH Results aQu K2801 Pin 30% DMSO 30% DMSO 3x SSC spot diameter 360 µm 30% DMSO 1.5x SSC, spot diameter 90 µm 30% DMSO 150 mM PP, 0.1% SDS 30% DMSO 150 mM PP, 0.05% SDS 30% DMSO 150 mM PP 30% DMSO 30% DMSO with 1.5x SSC gives good results, further SSC- concentrations will be tested
- 10. November 2004DMSO Spotting BufferMicroarray Solution SCHOTT Jenaer Glas GmbH Results aQu K2801 Pin 25% DMSO 0.005% SDS 50% DMSO 1.5x SSC, 0.005% SDS, spot diameter 110 µm 25% DMSO 1.5xSSC, 0.005% SDS Spot diameter 160 µm 50% DMSO 75 mM PP best results were obtained when combining DMSO with SSC and with or without SDS (but only for f.c. SDS of 0.005%, higher concentrations lead to precipitation) a combination of DMSO, phosphate buffer and SDS does not work further tests with additional combinations of DMSO, SSC and other detergents will be tested
- 11. November 2004DMSO Spotting BufferMicroarray Solution SCHOTT Jenaer Glas GmbH Results aQu K2801 Pin 50% DMSO 1.5x SSC, 0.025% CTAB 50% DMSO 1.5x SSC, 0.025% Triton X100 50% DMSO 2.5x SSC 50% DMSO 2x SSC best results were obtained when combining DMSO with SSC and with or without detergents spot sizes range between 100 and 140 µm similar results were obtained with 30% DMSO 50% DMSO 1.5x SSC, 0.005% SDS
- 12. November 2004DMSO Spotting BufferMicroarray Solution SCHOTT Jenaer Glas GmbH Summary 50 (30%) DMSO in water usually gives very small spots on Slide E (approximately 60 µm) addition of SSC (f.c. 1.5x to 2.5x) with or without detergent (f.c. 0.005% to 0.025%) gives good spot morphology and size spot size can be increased by increasing SSC concentration and/or adding detergent 50(30)% DMSO is not compatible with Next Spot or phosphate buffer (150 mM f.c. phosphate buffer), addition of detergent does not improve spot morphology*
Notas del editor
- The complete labeling protocol as pdf is available upon request Lot-Numbers of dyes used: Dye 3E08-03158 Dye 5E06-04115 Cy3311839 Cy5311840 Alexa 55502E2-1 Alexa 64702E1-1
- spotting buffers from second test all combinations of DMSO, SSC and 0.05% or 0.1% SDS precipitated and could not be tested
- spotting buffers from second test all combinations of DMSO, SSC and 0.05% or 0.1% SDS precipitated and could not be tested
- selected spotting buffers from first test
- selected spotting buffers from first test CTAB = cetyl- trimethylammonium bromide
- spotting buffers from second test all combinations of DMSO, SSC and 0.05% or 0.1% SDS precipitated and could not be tested
- spotting buffers from second test all combinations of DMSO, SSC and 0.05% or 0.1% SDS precipitated and could not be tested
- selected spotting buffers from first test
- selected spotting buffers from first test CTAB = cetyl- trimethylammonium bromide
- * further tests are ongoing