Alk-Phos Direct Labeling

Dominik Haudenschild, June 20, 1998

Solutions:

Hybridization Buffer:
Calculate volume of hyb buffer needed
Add NaCl to 0.5M
Add Blocking reagent to 4%, stir 1-2 hours to dissolve
-Store at -20° to 6 months

Primary Wash Buffer (1L)
2M Urea 120g
0.1% SDS 1g
50mM Na-Phosphate 100ml 0.5M pH 7.0 stock
150mM NaCl 8.7g
10mM MgCl2 10ml 1.0M stock
-Store at 4° to 1 week

Secondary Wash Buffer 20x stock (1L)
1M Tris 121g
2M NaCl 112g
pH to 10.0
-Store at 4° to 4 months

Secondary Wash Buffer - Working Dilution
 Make 1x, then add 1M MgCL2 to 2mM (2ml/L)
-Use immediately, do not store

Volumes needed:
 Small blots @ 90 cm2 = 7 ml Hyb Buffer
use 140ng probe for low copy number
use 35ng probe for high copy number
 Large blots @ 300 cm2 = 20 ml Hyb Buffer
use 600ng probe for low copy number
use 150ng probe for high copy number

 

Labeling 150ng (600ng) Probe:
  1. Dilute 20µl cross-linker solution with 80µl water
  2. Dilute DNA to 10ng/µl
  3. Place 15µl (60µl) of DNA in 0.65ml tube and boil 5 minutes
  4. Snap-cool DNA on ice for 5 minutes, keep DNA and all reagents on ice
  5. Add 15µl (60µl) reaction buffer, mix gently but thoroughly
  6. Add 3µl (12µl) labeling reagent, mix gently but thoroughly
  7. Add 15µl (60µl) diluted cross-linker solution. Mix thoroughly, spin to collect
  8. Incubate 30 minutes at 37°C
  9. Use probe immediately or
    Store on ice up to 2 hours or
    Add 50µl (135µl) glycerol, then store -20° up to 6 months

Hybridization and Washes:
  1. Preheat all solutions to desired temperatures before use
  2. Pre-hyb for at least 15 minutes at 55°C
  3. All probe to pre-hyb buffer
  4. Hybridize overnight at 55°C (2-4 hours is ok for med. to hi target levels
  5. Wash two times 10 minutes in Primary Wash Buffer preheated to 55°C
  6. Wash two times 5 minutes in Secondary Wash Buffer at Room Temperature
    no more than 30 minutes!

Detection with ECF:
  1. Use 25µl/cm2 of ECF substrate = 2.3ml (9.3ml)
  2. Drip-dry excess wash buffer from blots, place sample-side up onto clean surface
  3. Pipette ECF onto surface of blot, spread with 5ml pipette, incubate 1 minutes
  4. Place into detection bag, seal, and incubate RT for 1 to 24 hours (keep out of light)
  5. Place bag face down onto Storm. Ethanol/water on glass plate ensures better contact
  6. Scan. Excitation wavelength = 430nm, Emission wavelength = 560nm