ECL RANDOM PRIME LABELLING AND DETECTION METHOD

The Amersham protocol with additions from the T. brucei Genome Project... courtesy of Vanessa Leach and Sara Melville

GeneImages random prime labelling module

• RPN 3540 30 reactions 
  RPN 3541 60 reactions

Dilute 50-100 ng DNA to be labelled to a volume of 20 µl in 0.5 ml PCR tube.
Overlay with oil.
Denature by boiling for 5 min (N.B. must be actively boiling, or 99.9°C on PCR block) then cooling on ice for 5 min.
Add the following reagents in order:

• 14 µl H2O
  10 µl Nucleotide mix
  5 µl Primers
  1 µl Klenow

  50 µl total

Mix gently and spin briefly in a microcentrifuge to collect contents at the bottom of the tube.
Incubate at 37°C for 1 hour.
Terminate the reaction by adding EDTA to a final concentration of 20 mM (5 µl of 0.2M EDTA).
N.B. The probe can be stored at -20°C in the dark for at least 12 months.

Amount of template per labelling reaction (ng)	50	100	200	500	1000	2000
Amount of fluorescein labelled probe (synthesized) after 1 hour at 37°C (ng)	315	340	455	510	580	705

Prepare the hybridization buffer as follows:
5X SSC
0.1% (w/v) SDS
5% Dextran sulphate
10% (v/v) liquid block (supplied in kit)
100 µg/ml denatured sheared heterologous DNA

Make up to the required volume. Gentle heating and constant stirring is required to completely dissolve the dextran sulphate. (Make up a stock volume of this buffer (minus ss DNA) and store -20°C.)

• Preheat the required volume of hybridization buffer (0.125 ml/cm2) to 65°C then place the blots in the buffer and prehybridize for 1 hour at 65°C with constant gentle agitation.
  • N.B. if the filters are new, rinse them in 5X SSC at 65°C for 30 min - 1 hour before prehybridization and also discard the prehybridisation buffer and replace with fresh hybridisation buffer before adding the probe. This will get rid of some colony debris and reduce background.
  • Excess hybridization buffer can be stored at -20°C for at least 12 months.
• Remove the required amount of probe to a clean microcentrifuge tube (to give concentration of 1-5 ng/ml).
• Denature probe by boiling for 5 min (or on PCR block at 99.9°C) then cooling on ice.
• Centrifuge briefly then add to the prehybridization buffer, avoiding placing it directly on the blots, and mix gently.
• Hybridize for 2 hours at 65°C with gentle agitation.
• Preheat wash solutions to 65°C.
• Pour off hybridization buffer and briefly rinse filters in 5X SSC at RT .
• Wash filters in excess 1X SSC, 0.1% (w/v) SDS for 15min at 65°C .
• Pour off wash solution and replace with excess 0.5X SSC, 0.1% (w/v) SDS. Wash for 15 min at 65°C .

ECL Detection Reagents RPN 3004 (2000 cm2)

The following steps are all performed at room temperature and all the incubations require constant agitation of the filters.

Buffer A: 100mM Tris-HCl, 600mM NaCl, pH7.5

• Place the filters in a clean container and rinse with buffer A for 1 min.
  • Use 2ml of buffer for each cm2 of membrane.
• Discard the wash solution and replace with 10% (v/v) liquid block in buffer A.
• Incubate for 30 min.
• Dilute the anti-fluorescein-HRP conjugate 1000-fold in freshly prepared 0.5% (w/v) BSA Fraction V in buffer A.
  • The amount prepared should be equivalent to 0.25 ml/cm2 of membrane.
• Place the filters in an appropriate sized container (of similar size to the filter) and incubate in diluted conjugate solution for 30 min.
• Place the filters in a clean container and remove unbound conjugate by washing for 3 x 10 min in excess 0.1% (v/v) Tween-20 in buffer A.

4. SIGNAL GENERATION AND DETECTION

• Mix an equal volume of detection solution 1 with detection solution 2 to give sufficient to cover the filter. 0.125 ml/cm2 is recommended.
• Drain the excess reagent from the washed filters and place them in a clean container.
• Add the detection reagent directly to the filter on the side carrying the DNA.
• Incubate for 1 min at room temperature with gentle agitation.
• Drain off the excess detection regent and wrap filter in SaranWrap.
• Gently smooth out air pockets.
  Place the filter, DNA side up, in the film cassette. In the dark, place a sheet of autoradiography film on top of the filter and close the cassette.
• Expose the film for the required length of time (1 min - overnight) and develop the film.

This method has been taken from the protocol book supplied with Amersham's ECL random prime labelling and detection kit, version II. The alterations I have made are indicated in the notes and highlighted in bold.

The adapations are due to high background on the P1 library filters produced by specific colonies in the P1 library with endogenous peroxidase activity. The detection reagents in the kit are sensitive to this activity so it is necessary to alter the hybridization and blocking conditions to block this activity. The specific changes are summarised below:
1. Wherever the liquid block appears in the protocol, use 10% block not 5%.
2. Prehybridization, hybridization and washes are carried out at65°C not 60°C.
3. If there is high background reduce the concentration of probe in the hybridization. However, this depends on the individual probe.
4. If the filter has not been used before it is useful to wash off as much of the colony debris as possible. This may be done by rinsing the filter in 5X SSC at 65°C and by changing the prehybridization buffer before hybridization.

Vanessa Leech

mildly adapted by Mark Blaxter (very mildly)