Equipment

NanoDrop (from VLP)

 * Open program
 * Login with username:Khavari, password:mouse
 * To measure DNA:
 * Click on “nucleic acids” button
 * Wipe down reader w/ dry kimwipe
 * Add water to clean reader—push pedestal up and down click a couple times
 * Wipe down with kimwipe
 * Add drop of “blank” and click measure to clean machine
 * Add 2ul of “blank” click on “blank” button to black reader
 * Clean blank from reader, clean with water as above
 * Add 2ul sample (undiluted) to reader
 * Push down pedestal gently
 * Click the “measure” button
 * Machine will click a couple times and measure sample—label sample in “sample ID” block
 * Clean reader with wipe
 * Add water to clean reader between samples
 * Repeat to measure next sample

Cryostat
(from Katie)
 * Set machine at -25°C
 * Never touch the snowflake button=defrost machine!
 * Set slicer @ 7mm (controlled by knob at top right)—typical slice size needed for ADS
 * Put in blade (sharp blade!)—lock down with lever on right
 * ​Change blade each time machine is used
 * Place sample holder in cooling location—do NOT jam into hole—will get stuck
 * Add dime-sized amount of OCT to circular part
 * Place sample in center
 * Let cool (OCT will change color—clear to white)
 * Place glass slide in holder (located in lab by enzymes)
 * ​Use clean, smooth edge towards blade
 * Align with a 1-2 mm difference between blade and glass slide edges (glass below blade)
 * Lock in glass slide with lever
 * Move glass slide to “open”—facing left
 * Only have glass facing blade when going down with spool
 * ​Instead of using glass slide, can use brush to hold section and gently pull as you move the lever
 * Place cooled sample lengthwise on sample holder with sample coming at you and slight angled backwards
 * Turn spinning wheel to move sample back and forth—start with sample far back and slowly move forward with control buttons on left side until it starts slicing
 * Return glass slide to blade position
 * Spin wheel once to get sample slice
 * Move glass slide back to left position to free sample, use brush to smooth and pick up with slide (place 2 slices per slide closely together, ~10 slides per sample—or dependent on experiment)
 * Use only Polylysine-coated slides (found in green and yellow box)
 * Place sample on “writing” side of slide
 * Stain sample with hematoxylin quickly to determine sample quality
 * Rinse quickly in water, dry a bit, and check under microscope
 * Once done slicing, warm sample by placing holder at rt—label slides while warming sample.
 * Attach next sample to holder w/OTC and cool
 * Once warm, cut sample off with razorblade, cut off excess materials, return to block and store in bag—place on DRY ICE
 * Store slides at -20°C—DO NOT FREEZE/THAW before staining