Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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The material on this page is part of Chapter 10, which is shown in full as a preview on this site.

Chapter 10: Nucleic Acid Platform Technologies

Rando Oliver, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

INTRODUCTION

Printing Microarrays

(Protocol summary only for purposes of this preview site)

Most laboratories will choose to order microarrays from a commercial vendor, such as NimbleGen, Agilent, Affymetrix, or Illumina. These vendors sell a number of products encompassing genomes from the three domains of life. In addition, custom microarrays can be made to order by most of these companies. Microarray printing, however, is sufficiently straightforward that even small laboratories with extensive microarray needs may find it cost-effective and worth the effort to produce their own microarrays. Printing microarrays requires a spotting robot. Several commercial spotting robots are available, such as the OmniGrid series (Digilab Genomic Solutions). These instruments are expensive and will most frequently be housed in institutional core facilities or in large laboratories. As an alternative, designs for building a spotting robot have been available from the Brown laboratory at Stanford for many years. Protocols for building a spotting robot are beyond the scope of this chapter, but interested parties can find a protocol at http://cmgm.stanford.edu/pbrown/mguide/.


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 Protocol 1: Printing Microarrays

Most laboratories will choose to order microarrays from a commercial vendor, such as NimbleGen, Agilent, Affymetrix, or Illumina. These vendors sell a number of products encompassing genomes from the three domains of life. In addition, custom microarrays can be made to order by most of these companies. Microarray printing, however, is sufficiently straightforward that even small laboratories with extensive microarray needs may find it cost-effective and worth the effort to produce their own microarrays. Printing microarrays requires a spotting robot. Several commercial spotting robots are available, such as the OmniGrid series (Digilab Genomic Solutions). These instruments are expensive and will most frequently be housed in institutional core facilities or in large laboratories. As an alternative, designs for building a spotting robot have been available from the Brown laboratory at Stanford for many years. Protocols for building a spotting robot are beyond the scope of this chapter, but interested parties can find a protocol at http://cmgm.stanford.edu/pbrown/mguide/.

For typical microarrays, oligonucleotides are prepared at 2040 M in aqueous 3 SSC and are distributed into 384-well plates. Oligonucleotides are printed to polylysine-coated microarray slides. It is best to print when the humidity is at least 50. When the humidity is 2030, there can be problems with pins drying. To increase or maintain proper humidity, consider building a humidity-controlled enclosure around the robot. For robots lacking an enclosure, it may be adequate to run a humidifier or two during the printing procedure.

The details of a printing protocol vary depending on the robot being used; thus, follow the manufacturer's instructions. However, a typical workflow using a robot equipped with contact steel quill pins is provided in this protocol.


MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

Recipes for reagents specific to this protocol, marked <R>, are provided at the end of the protocol. See Appendix 1 for recipes for commonly used stock solutions, buffers, and reagents, marked <A>. Dilute stock solutions to the appropriate concentrations.

Reagents

  • Oligonucleotides of desired sequences
    • Oligonucleotides are typically prepared at 2040 m in aqueous 3 SSC.
  • Salmon sperm DNA <A>, sheared (150 ng/L resuspended in 3 SSC)
  • SSC (3) <A>

Equipment

  • Desiccator
  • Nitrogen gas, compressed
  • Plates (384 wells)
  • Polylysine-coated slides
    • Although polylysine coating can be done in-house, in our experience, the failure rate is significant and purchasing commercial polylysine-coated slides (e.g., Erie Scientific) has proven to be the most cost-effective solution.
  • Slide box, plastic
  • Spotting robot


METHOD
Preparing to Print
Day 0

  • 1. Array the oligonucleotides into 384-well plates with identical volumes per well (typically 1020 L per well). If the oligonucleotide solutions were frozen, thaw the master plates overnight at 4C. If the oligonucleotides were dried, resuspend them in 3 SSC, and let them incubate overnight at 4C.
  • 2. Test that the spotting pins are all printing and that the arrayer is properly calibrated by performing test prints (Fig. 1). To closely approximate the viscosity of the actual print plates, use a test print plate consisting of 150 ng/L sheared salmon sperm DNA resuspended in 3 SSC. All pins should be able to print several hundred consecutive spots. In addition, test print in several locations on the slide platter to determine whether the platter has warped significantly since its last use.
    • If test prints indicate that the spotting robot is not performing as it should, see Troubleshooting.

Day 1

  • 3. Perform one more test print to make sure that the pins are still printing properly.
  • 4. Place slides gently onto the arrayer platter. Make sure that all slides are sitting flat and are firmly attached (by vacuum, clips, or tape, depending on the arrayer).
    • Handle the slides carefully to avoid producing minute glass chips, which might settle onto the surface of the slide.
  • 5. Blow dust off the slides with compressed nitrogen.
  • 6. If the arrayer has a sonicator water bath for cleaning the pins, fill it with fresh water.
  • 7. Transfer four 384-well oligonucleotide-containing print plates from 4C, and let them come to room temperature for at least 1 h.
  • 8. Centrifuge the plates at 1000 rpm for 2 min to remove condensation from the plate covers. Carefully remove a plate lid and the adhesive plate cover from one of the print plates.
    • Be careful not to jolt the plate because cross-contamination between wells will cause serious problems in any downstream data analysis.
  • 9. Place a plate into the spotting robot's plate holder.

Printing the Microarrays

  • 10. Start the print run. Keep careful notes about plate order and orientation. Knowing the plate order is necessary for creating the .gal file that maps spot position to gene name when microarray data are analyzed. Any mistakes during printing such as printing plates out of order (e.g., plate 3 before plate 2) can be corrected later on as long as they are noted.
  • 11. When a plate is finished printing and the print head has come to a complete stop, either let the plate evaporate in a hood if the plates are stored dry, or cover the plate with foil and its lid and store the plate at 80C.
  • 12. Insert the next print plate into the plate holder.
  • 13. When the print run is done, let the slides dry overnight, unless you are performing back-to-back print runs.

Day 2

  • 14. Transfer all slides from the arrayer into a plastic slide box. Store them in a desiccator.
  • 15. Power down the arrayer.


TROUBLESHOOTING

Problem (Step 2): Printing pins are not printing properly.

Solution: If any pins are not printing, four steps may be taken to improve performance. First, if dirt or other material has clogged the quill tip, wash the pins extensively in a sonicator bath. Second, if examination of a pin under a microscope indicates that the pin is clogged, try clearing the quill tip carefully with a razor blade, which may dislodge the contaminant. Third, subtle variation in pin length may prevent some pins from printing well; swapping pins within the print head may improve printing. Finally, if a pin continues to fail after all of the above steps have been taken, replace it with a fresh pin.

Problem (Step 2): Test plate printing is not uniform everywhere on the arrayer.

Solution: If the arrayer fails to print at particular locations, adjust the print height for that section of the platter. These adjustments can be made using the software that accompanies the robot.


DISCUSSION

A successful print run requires that print plates be handled carefully to prevent cross-contamination, that pins be checked regularly (i.e., after every 384-well plate or two) to confirm that they are still printing, and that the level of water in the bath (or sonicator) for washing pins be always full enough to cover the pins. We typically add 510 mL of water to the sonicator every 6 h during a print run, but this will depend on the ambient humidity.

To check that pins are still printing, shine a flashlight onto the slide at an angle. The salt deposited during printing is white, and when a pin stops printing the pattern of one sector deviates from the others. For example, if five rows of 20 spots have been printed, a perfect 520 rectangle can be seen, but if a pin has dropped out, then the rectangle will be incomplete.

If a pin stops printing, it may be possible to remove the pin carefully and clean it under a microscope if any dust is observed in or on the pin. If no dust can be seen, sometimes a pin can be restored by extensive sonication. Finally, replacing the faulty pin with a spare pin, or moving pins around in the print head, can be used as a last resort. After a print run has been paused, replace the first slide with a clean slide and perform another test print to check on the new arrangement. When restarting the spotting robot, be sure that printing recommences at exactly the location where the print run was paused.

WWW RESOURCE
Protocol for building a spotting robot http://cmgm.stanford.edu/pbrown/mguide/.

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