Fry lab: biology and management of Phytophthora infestans

1-2 ug DNA	x ul
spermadine (10 mg/ml)	2.0 ul
10X enzyme buffer	4.0 ul
Eco R1 enzyme	2.0 ul
RNase A (10 mg/ml)	1.0 ul
H2O	to 40 ul

4. Place the tubes in the 37° C incubator for at least 6 hr or overnight.

5. Add running dye to each sample, load onto a 0.8 % agarose gel and run overnight at 30-40 volts.

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Southern Blot protocol for use with radioactive and non-radioactive hybridization

(Modified from Sambrook et al. "Molecular Cloning, 1989")

1. Depurinate DNA by soaking gel in 0.25M HCl for 10 min with shaking at room temperature. Transfer the gel to a clean dish.

2. Denature DNA by soaking gel in 250-500 ml of 1X Southern base solution at room temp with shaking for 15 min. Pour off solution and soak gel in another volume of 1X Southern base solution.

3. Pour about 500 ml of 1X Southern base solution into a dish. Place a piece of glass or plastic across the dish to use as a support.

4. Place a piece of gel blot paper across the glass support, and allow it to hang into the base solution. This will act as a wick.

5. Place the gel on top of the wick, and roll it with a pipette to remove air bubbles.

6. Cut a piece of nylon membrane (about the same size as the gel) and soak it in 1X Southern base solution. We use Hybond N+ membrane. Place the membrane on top of the gel.

7. Cover the edge of the membrane to the edge of the dish with saran wrap on all four sides of the gel.

8. Place three pieces of gel blot paper on top of the membrane. Then put a stack of paper towels on top of the gel blot paper.

9. Finally, place a glass plate and a weight on top of the entire thing.

10. Let the DNA transfer for 3-6 hours. Then, remove membrane from gel and place in 200 ml 1M Tris pH 7.5, 1.5M NaCl and shake for 2-3 minutes to neutralize the base solution. Membrane can be allowed to dry or can be used immediately.

0.25 M HCl

21.55 ml concentrated HCl

Add the acid to 800 ml distilled water and adjust to 1 liter.

2X Southern Base Solution

20 g NaOH

175.25 g NaCl

Add H₂O to a total volume of 1 liter

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Hybridization with Renaissance non-radioactive kit

These instructions are basically identical to those which come with the kit (sold by NEN life science products catalog # NEL603)

1. Production of a fluorescein-labeled probe.

1. Thaw all labeling components except for the Klenow Fragment, and place on ice. Keep the Klenow at -20° C until just before use.
2. Briefly microfuge all kit components
3. Place 250 ng RG57 template DNA in a clean screw cap microfuge tube and dilute with water to a final volume of 19 ul. Put the tube in a 95-100° C heat block for 3-5 min, then quickly place the tube on ice for 5 min.
4. Add the following to the microfuge tube containing 19 ul denatured DNA:

Random primers and reaction buffer mix	5 ul
Fluorescein nucleotide mix	5 ul
Klenow fragment	1 ul
	Total volume 30 ul

5. Microfuge the reaction tube briefly and incubate for 1 hour at 37° C. For greater probe synthesis, incubate the reaction overnight at room temperature.
6. Terminate the reaction by adding 5 ul 0.1M EDT

2. Purification of fluorescein-labeled probes.

1. For each probe, use 1 Bio-Rad micro bio-spin column.
2. Shake the column several times to resuspend the gel and to remove bubbles.
3. Snap off the tip and place the column in a 2.0 ml microfuge tube. Remove the cap from the column. Let the column drip for about 2 min. and discard the drained buffer.
4. Centrifuge for 1.5 min at 7,000 in the small microfuge. Discard the drained buffer.
5. Add 50 ul TE buffer to your labeled RG57 probe (from step I). Pipette entire sample (85 ul) into the column. Centrifuge for 1.0 min at 7,000 in the small microfuge.
6. Add 100 ul TE buffer to the column, and microfuge for 1 min.
7. The probe is now finished and can be used immediately or stored at -20° C for 1 year.

3. DNA hybridization

Day 1

1. Incubate salmon sperm DNA (10 mg/ml) in a 95° C heat block for 5 min. Place immediately on ice for 5 min..
2. Wet the membrane containing the denatured target (digested P. infestans) DNA in 2X SSC.
3. Insert membrane into a hybridization tube and add prehybridization buffer containing 50 ug/ml salmon sperm DNA. Use 0.1 ml buffer /cm² of membrane. Our membrane is generally 300 cm² so we use 30 ml prehybridization buffer and 150 ul salmon sperm DNA (10 mg/ml).
4. Incubate at 65° C in a hybridization oven for at least 1 hour.
5. Combine the probe (from step II) with 200 ul hybridization buffer and 75 ul salmon sperm DNA. Heat at 95° C for 5 min and immediately place on ice for 5 min.
6. Add the denatured probe mix (from step 5) to 15 ml of hybridization buffer which has been warmed to 65° C.
7. Empty the prehybridization buffer from the tube (this can be dumped down the drain), and add the hybridization buffer containing the probe and salmon sperm DNA (from step 6).
8. Hybridize overnight in the oven at 65° C.

Day 2

1. Pour hybridization solution into a tube or bottle and place at -20° C. This solution can be used for a future hybridization.
2. Add 100 ml 2.0X SSC, 1.0% SDS to the tube and wash the membrane for 15 min at 65° C. Dump wash solution down the drain.
3. Add 100 ml 0.2X SSC, 0.1% SDS to the tube and wash for 15 min at 65° C. Dump wash solution down the drain.
4. Carefully remove membrane from tube and place in a clean plastic dish.

IV. Detection

1. All of the following steps are done at room temperature.
2. Add 300 ml detection buffer 1 to the membrane in a plastic dish and vigorously agitate the membrane for 5 min. We use a shaking platform.
3. Place the membrane in 30 ml detection buffer 2 for 1 hour with gentle agitation.
4. Add 30 ul antifluorescein-ap conjugate to 30 ml buffer 2, and place the membrane into this antibody conjugate solution. Incubate for 1 hour with gentle agitation.
5. Vigorously wash the membrane 4 X 5 min in detection buffer 1.
6. Vigorously wash the membrane 2 X 5 min in detection buffer 3.
7. Transfer the membrane from the final antibody conjugate wash solution to a clean container.  MAKE SURE THE MEMBRANE IS DNA SIDE UP.
8. Completely cover the membrane with CDP-Star. We use about 3 ml (0.01ml/cm² of membrane).
9. Incubate the substrate on the membrane for 5 min.
10. Gently remove excess solution with blotting paper.
11. Wrap the damp membrane with saran wrap.
12. Place the prepared membrane, DNA SIDE UP, in a film cassette and place reflection autoradiography film over the membrane.
13. Expose the film for 5 minutes, then develop.

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Solutions for non-radioactive detection

0.1 M EDTA pH 8.0

Add 3.68 g of disodium EDTA to 80ml of dH₂O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH. Bring to a final volume of 100 ml with dH₂O. Pass through a 0.22 uM filter.

20X SSC

Dissolve 175.4 g of NaCl and 88.2 g of sodium citrate in 800 ml of dH₂O. Adjust the volume to 1 liter with H₂O. Pass through a 0.22 uM filter.

DNA prehybridization and hybridization solution

2 X SSC

0.5% (w/v) blocking reagent

5% (w/v) dextran sulphate

0.1% (w/v) SDS

To make 100 ml:

Add 10 ml of 20 X SSC and 0.5 g blocking reagent to 50 ml of distilled H₂O. Before adding the dextran sulphate, heat gradually up to 60° C with continuous stirring to dissolve the blocking reagent. Slowly (over 2-3 min) add 5 g dextran sulphate and stir until dissolved. Add 0.5 ml of a 20% solution of SDS. Bring to a final volume of 100 ml with dH₂O. Store at -20° C when not in use.

Salmon sperm DNA

100 mg Salmon sperm DNA

10 ml H₂O

Stir for 1-2 hours until the DNA is dissolved

To sheer the DNA autoclave for 10 minutes.   Store in aliquots at -20 C.

Hybridization wash 1

2 X SSC, 1.0% SDS

100 ml of 20 X SSC

10 g of SDS

Add water to a total volume of 1 liter

Hybridization wash 2

0.2 X SSC, 0.1% SDS

10 ml of 20 X SSC

1 g of SDS

Add water to a total volume of 1 liter

Detection buffer 1

0.1M Tris, pH 7.5

0.15M NaCl

To make 1.0 Liter:

Combine 12.2 g Tris and 9.0 g NaCl in 500 ml dH₂O. Adjust pH to 7.5 with HCl while stirring. Bring to a final volume of 1.0 liter with dH₂O. Pass through a 0.22 uM filter. Store at room temperature.

Detection buffer 2

0.1M Tris, pH 7.5

0.15M NaCl

0.5% (w/v) blocking reagent

To make 100 ml:

Comebine 0.5 g of blocking powder with 100 ml detection buffer 1. Heat gradually up to 60° C with continuous stirring to dissolve the blocking reagent. Store at -20° C when not in use.

Detection buffer 3

0.1M Tris, pH9.5

0.2M NaCl

To make 100 ml:

Combine 1.21 g of Tris and 0.59 g of NaCl with 75 ml of dH₂O. Adjust pH to 9.5 with HCl. Bring to a final volume of 100 ml with dH₂O. Pass through a 0.22 uM filter. Store at room temp.

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Radioactive random-primer labeling Southern hybridization protocol

1. Production of a ³²P-labeled probe.

1. Thaw all labeling components except for the Klenow Fragment, and place on ice. Keep the Klenow at -20° C until just before use. We use the BMB random primer labeling kit.
2. Place 50 ng RG57 template DNA in a clean screw cap microfuge tube and dilute with water to a final volume of 14 ul. Put the tube in a 95-100° C heat block for 3-5 min, then quickly place the tube on ice for 5 min.
2. Add the following to the microfuge tube containing 14 ul denatured DNA:

Random primers (from kit)	2.5 ul
Random primer buffer mix (from kit)	2.5 ul
Add the following behind a shield
32P-labeled dCTP	5.0 ul
Klenow fragment	1.0 ul  Add the Klenow fragment last
Total volume	25 ul
Incubate for about 1 hour at 37° C.

2. Purification of ³²P-labeled probes.

1. For each probe, use 1 Bio-Rad micro bio-spin column.
2. Shake the column several times to resuspend the gel and to remove bubbles.
3. Snap off the tip and place the column in a 2.0 ml microfuge tube. Remove the cap from the column. Let the column drip for about 2 min. and discard the drained buffer.
4. Centrifuge for 1.5 min at 7,000 in the small microfuge. Discard the drained buffer.

Do all of the following steps behind a shield

5. Add 50 ul TE buffer to your labeled RG57 probe (from step I). Pipette entire sample (85 ul) into the column. Centrifuge for 1.0 min at 7,000 in the small microfuge.
6. Add 100 ul TE buffer to the column, and microfuge for 1 min.

3. Prehybridization

1. Make up enough hybridization solution for your experiment (We use about 25 ml per blot).
2. Insert membrane into a hybridization tube and add hybridization buffer.
3. Incubate at 65° C in the hybridization oven for about 1 hour.

    IV. Hybridization

Do all of the following behind a shield

1. Heat your probe at 95° C for 5 min (to denature it) and immediately place on ice for 5 min.
2. Add the denatured probe mix directly to the hybridization tube.
3. Hybridize overnight in the oven at 65° C.
4. Pour hybridization solution in liquid radioactive waste.
5. Add 50 ml 2.0X SSC, 0.1% SDS to the tube and wash the membrane for 15 min at 65° C. Pour off wash solution into radioactive waste.
6. Add 50 ml 0.5X SSC, 0.1% SDS to the tube and wash for 15 min at 65° C. Pour off wash solution into radioactive waste.
7. Repeat step 6.
8. Carefully remove membrane from tube and wrap with saran wrap.
9. Place membrane in a film cassette in contact with X-ray film. Expose overnight.

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Solutions for Radioactive Detection

50X Denhardt’s solution

5 g Ficoll (Type 400, Pharmacia)

5 g polyvinylpyrrolidone

5 g bovine serum albumin (Fraction V; Sigma)

Add H₂O to a total volume of 500 ml.

20X SSC

Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of H₂O. Adjust the pH to 7.0 with a few drops of a 10 N solution of NaOH. Adjust the volume to 1 liter with H₂O. Dispense into aliquots. Sterilize by autoclaving.

0.5 M NaPO₄ buffer pH 7.0

577 ml of 1M Na₂HPO₄

423 ml of 1 M NaH₂PO₄

Hybridization buffer (25 ml)

2.5 ml of 50 X Denhardt’s solution

6.25 ml of 20 X SSC

2.5 ml of 0.5 M NaPO₄ buffer pH 7.0

0.5 ml of 0.5 M EDTA

12.63 ml H₂O

0.625 ml of 20% SDS

2 X SSC, 0.1% SDS

100 ml of 20 X SSC

1 g of SDS

Add water to a total volume of 1 liter

0.5 X SSC, 0.1% SDS

25 ml of 20 X SSC

1 g of SDS

Add water to a total volume of 1 liter

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Late blight rating system