Rhodamine Marker Spot Protocol

 

Introduction

In order to photograph the location of spots on the array, "marker" spots must be printed in a 4 point "frame" around the edges of the microarray. The marker consists of Rhodamine dye in a mixture of methyl salicylate and PDLA (a lactic acid polymer) and is prepared as follows.

Materials for Microarray Printing
Item
Product Number, Supplier
Use
Rhodamine B
R-6626, Sigma
Printed on arrays as fluorescent reference point
poly-d, l- lactic acid (PDLA)
16585, Polysciences
Polymer that sequesters Rhodamine B
Methyl Salicylate
M-2047, Sigma
Solvent for PDLA and Rhodamine B

Protocol

100 mg/ml PDLA Solution

  1. Measure 100 mg of poly d,l lactic acid (PDLA) and place in a 1 mL eppindorf tube.
  2. Add 1 ml of methyl salicylate to tube and vortex for 30 minutes on highest setting.
  3. Invert tube to ensure that the polymer is properly mixed. Vortex more if needed.

8 uM Rhodamine Solution

  1. Measure out 38.3 mg of Rhodamine.
  2. Add Rhodamine to 1 mL eppindorf tube.
  3. Add 1 mL of methyl salicylate to Rhodamine and vortex until dye is completely suspended.
  4. The Rhodamine is now at a concentration of 0.08 M.
  5. Dilute an appropriate amount of this solution with methyl salicylate to achieve a Rhodamine solution of 8 uM (two 100 fold dilutions of the 0.08 M solution will give an 8 uM solution).

Combine Solutions

  1. Add 500 ul of 8 uM Rhodamine to 500 ul of 100 mg/ml PDLA solution to achieve a final concentration of 4 uM Rhodamine.
  2. Aliquot into eppindorf tubes and store at -20 degrees C.

Printing Marker Spots

  1. Using an array pin that is only for printing marker spots, print a "frame" of 4 spots around the 4 corners of the microarray.
  2. Ensure that the pin cleaning protocol is sufficient to remove the PDLA from the pins.