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NSF / U of Illinois Microarray Workshop - Steve Clough / Vodkin Lab

NSF / U of Illinois Microarray Workshop - Steve Clough / Vodkin Lab. Troubleshooting. The Good. The Ugly. The Bad. Spot size uniformity. Low Salt. High Salt. NSF / U of Illinois Microarray Workshop - Steve Clough / Vodkin Lab. Related to hydrophobic and ionic interactions between

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NSF / U of Illinois Microarray Workshop - Steve Clough / Vodkin Lab

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  1. NSF / U of Illinois Microarray Workshop -Steve Clough / Vodkin Lab Troubleshooting The Good The Ugly The Bad

  2. Spot size uniformity Low Salt High Salt NSF / U of Illinois Microarray Workshop -Steve Clough / Vodkin Lab Related to hydrophobic and ionic interactions between DNA suspension and surface coating. Example, salt concentration.

  3. NSF / U of Illinois Microarray Workshop -Steve Clough / Vodkin Lab Black Holes and Random Background Solutions: Wash slide with SDS prior to chemical blocking step. Add ‘cold’ polyA (or polyT) and/or, tRNA, calf thymus, etc. Use a column-based purification kit (ie: Qiagen PCR cleanup) to clean-up probe after labelling. Use lower laser and/or PMT settings on scanner.

  4. NSF / U of Illinois Microarray Workshop -Steve Clough / Vodkin Lab Probe Drying and/or Precipitating Hybridization solution dried under coverslip at edges Solutions: Ensure adequate humidity in hyb chamber. One can use moistened filter paper at ends of slide away from cover slip. Use coverslips (like LifterSlips) that are slightly raised to allow use of greater volume of hybridization solution.

  5. NSF / U of Illinois Microarray Workshop -Steve Clough / Vodkin Lab Smeared spots (‘comets’) Possible cause DNA spots smear across slide prior to adequate blocking of slide coating Solution Make first blocking treatment a vigorous wash in 0.2% SDS

  6. NSF / U of Illinois Microarray Workshop -Steve Clough / Vodkin Lab Cracking and Flaking of PL Coating Due to old or poorly made poly-L-lysine (PL) slides Solution: Spot, UV-link, and hyb PL slides within 3-4 months. Test a few slides of each PL batch before investing too much time, money, and resources

  7. NSF / U of Illinois Microarray Workshop -Steve Clough / Vodkin Lab Smeared spots within one grid Due to clogged/damaged pin tip

  8. NSF / U of Illinois Microarray Workshop -Steve Clough / Vodkin Lab Thanks to those that have helped us Shauna Sommerville’s Lab: Iian Wilson Erico Miura Shu-hsing Wu Pat Brown’s Lab: Camilla Mei-Fang Kao Pam Green’s Lab: Jeff Landgraf Matt Larson U of Illinois Keck Center Mark Band Jose Pardinas

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