Gelatin Preparation, DNA Purification, Sample Preparation and Microarray Printing

 

Materials

 

Preparation of 0.2% (w/v) Gelatin Solution

  1. Dissolve gelatin powder in sterile MilliQ water by gently swirling mixture for 15 minutes in a 60ºC water bath.
  2. Cool the 0.2% gelatin solution at room temperature, and, while still warm (~37-40ºC), filter it through a 0.45 µm cellular acetate membrane (CA). We usually prepare 100 ml at a time and store 50 ml aliquots of the filtered gelatin solution at 4ºC.

 

Notes on DNA Purification

DNA can be purified via any method that gives supercoiled plasmid DNA with a 260/280 absorbance ratio greater than 1.7. To purify many plasmids in parallel, we use the Qiagen Turbo Miniprep Kit. We seed bacterial clones in 1.3 ml of terrific broth (TB) in a 96-Deep-Well Plate and shake them at 250 rpm for 18-24 hours at 37ºC. Of course, DNA of equivalent or better quality can be purified via other methods and is also suitable for making transfected cell microarrays.

 

Preparation of Samples to Print

To prepare samples for printing, in a 96-well plate dilute purified plasmid DNAs with 0.2% gelatin to a final DNA concentration of 0.030-0.040 µg/µl. Make sure the final gelatin concentration remains greater than 0.17%. This requires DNA solutions of relatively high concentration. After dilution we mix the DNA-gelatin samples by pipetting and transfer them to a 384-well plate for printing with a robotic arrayer.

 

Notes on Microarray Printing

We use a PixSys5500 Robotic Arrayer with Telechem's ArrayIt Stealth Pins (SMP4), but have also successfully tested other arrayers. We program the arrayer to transfer the gelatin-DNA solution to the slide by touching the pins to the slide surface for 25-50 ms. Using SMP4 pins, the printed spots have diameters of 100-120 µm and we normally place them 300-400 µm apart. We always maintain a 55% relative humidity environment during the arraying and perform a thorough wash step between each dip into a DNA-containing sample. The dried spots on the slide are very difficult to see by eye because they contain low amounts of salt. To the right is a photograph of a dried 14 X 10 array that was taken with a light microscope. We have transfected slides as soon as one hour after printing, but we normally keep them for at least 16 hours before using them.

 

How to Store Printed Slides

We store printed slides at 4°C or at room temperature (~20-25ºC) in a vacuum desiccator containing anhydrous calcium sulfate pellets. We have not observed a detectable deterioration in performance after storage for greater than 3 months. See Tips and Information for other information on storage conditions.

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*Table of Contents *Sabatini Lab *Whitehead Institute for Biomedical Research*