Roger Wagner MD, PhD

July 2003

 

 

Post-processing GAPS II slides

 

  1. Place slides DNA side down between 2 tube racks and etch back of slide with diamond tip pen to mark edges of array.
  2. Place 75ml H20 or 2X SSC in Sigma humid chamber at room temp with table lamp 6 inches above.
  3. Place slides above liquid, DNA side down, for 2 minutes until spots glisten.
  4. Snap dry slide on inverted 90C heat block for 5 seconds.
  5. Crosslink DNA to slide using Stratagene UV Stratalinker set at 3000microjoules, ”Energy”, press start.

 

  1. Prehybridize arrays in prehyb mix at 42C for 60 min.  We use the Corning slide carriers from the microarray facility for prehyb.

 

Prehyb mix                                                           For 500 ml

5X SSC                                                                125ml 20X SSC

50% formamide                                                  250 ml formamide

0.1% SDS                                                            5 ml 10% SDS

0.1mg/ml BSA                                                     500 ul 10% BSA

                                                                              120 ml H2O

 

 

  1. Place arrays in metal slide rack and wash in Millipore H2O 2 min. X 2 at room temp.
  2. Wash arrays in 98C Millipore H2O (just stopped boiling)  2 min.
  3. Rinse arrays in isopopanol 1 min room temp.
  4.  Centrifuge arrays 700 rpm X 5 min. at room temp. to dry.

 

 

Necessary equipment:

Humid chamber

4

Sigma #H 6644

Inverted heat block (90C)

1

 

Diamond scriber

1

VWR #52865-005

Slide rack

2

Shandon Lipshaw #121

Slide chamber

2

Shandon Lipshaw #121

 

The GAPS II slides seem to be slightly heavier than regular slides, perhaps due to the array label.  It is important to weigh the arrays in the carrier in which they will be spun prior to processing in order to prepare an appropriate balance carrier for centrifugation.