Workshop
Protocols

Soybean
EST Project

Reference: Derived from Pat Brown lab home page

Need:

• 120 25x75x1 microscope slides
• 4 metal racks (capacity=30 slides each)
• 3 Rubbermaid containers: 7.8 Liter, 0008, #8 (13x8.5x5")
• 1 Rubbermaid containers: 1.7 Liter, 0166, 9" sq #12 (8x4.5x3.5")
• 1 small autoclave tub

1. Place slides in racks and racks into small Rubbermaid dish.
   Place this dish into autoclave tub (so spills from the small tub fall into larger tub)
   
   
2. Pour 2L 3.57M NaOH in 54.3% EtOH
   Cover with plastic wrap or lunch tray.
   Soak 2 hr on orbital shaker with gentle shaking.
   
   
3. Carefully transfer to NanoPure water rinse (3 spearate Rubbermaid tubs each holding several liters.
   Leave slides in the water while prepare poly-lysine solution.
   
   
4. Prepare 1.8 liter 0.01 % poly-L-lysine, 0.1X PBS solution in plastic beaker for each rack of slides (metal slide racks that hold 30 slides max)
   180 mls 0.1% Poly-L-Lysine (Sigma #P8920)
   180 mls 1X PBS
   1440 mls NanoPure water
   
   
5. Add 1.8 liter 0.01 % poly-L-lysine, 0.1X PBS solution to a Rubbermaid container.
   Transfer in the slides (in metal slide rack) from last water.
   Shake 60 minutes (speed #2 on Vodkin's Bellco Orbital Shaker).
   
   
6. Dip directly into a NanoPure water rinse (several liters) to rinse thoroughly.
   
   
7. Centrifuge slides on microtiter plate carriers (place paper towels below rack to absorb liquid) for 5 min. @ 500 rpm.
   
   
8. Finish drying for 10-15 minutes in 45c oven
   
   
9. Store slides at room temp in closed slide box (plastic only, without rubber mat bottom).
   
   Slides should be clean and transparent.
   Verify coating by scanning at high power.
   
   

** Brown Lab suggests to wait about 1-2 weeks prior to use in printing because slides become more hydrophobic the longer they sit in air (I presume it's due to more NH₃+ of the PL finding SiO- groups of the glass). Water should bead off (when slides are fresh, it doesn't). Slides tend to bind less DNA after about 3-4 months. Test print, hyb, and scan a given slide to test the batch prior to large scale use.

3.57M NaOH in 54.3% EtOH (2 liter, enough for 4 metal slide racks)

285.72 g NaOH in 850 mls H20
Stir to completely dissolve.
Add 1150 mls 95% EtOH (1090 ml 100% plus 60 ml water)
Stir until mixed-add more water if necessary to clarify solution. Will probably need about 100-150 mls to get all the alcohol to mix into the aqueous phase (cloudiness is due to phases not mixing-will see the phases separate if stop stirring)

Maniatis recipe for 1X PBS

8g	NaCl
0.2g	KCl
1.44g	Na2HPO4
0.24g	KH2PO4
800mls	H2O

pH to 7.4 with HCl
Add H₂O to 1000mls
Filter sterilize (also removes lint)