Method: Screening YAC Libraries

November 15, 1990

Jennifer Korte

Principle:

This is a method for screening YAC libraries for specific DNA sequences. Transformants are patched onto selective medium plates and colonies lifted to nylon filters for further growth. Cells are then lysed in place and the DNA is fixed to the filters. DNA probes are hybridized to the filters to test for the presence of specific sequences. A secondary screen is performed to ensure a positive hybridization.

Time required:

10 days

Day 1: 1-3 hours
Day 2: 2 hours
Day 3: 1 hour
Day 4: 1-2 hours
Day 5: 1-2 hours
Day 6: 30 minutes
Day 9: 1 hour
Day 10: 1 hour

Special Equipment and Supplies:

• Hybond-N 82 mm round nylon filters (Amersham #RPN.82N)
• 150 mm petri dishes

Special Reagents:

• SCE solution
• Dithiothreitol (DTT)
• Lyticase (Sigma # L8137)

Day 1

1. Using autoclaved toothpicks, patch isolated colonies onto gridded selective media plates appropriate to the vector used (i.e. AHC + tryptophan, use 4 ml of 1% trp / 1liter of media.) Include patches of a clone known to strongly hybridize to the probe of interest as well as a clone that does not hybridize to the same probe. These will serve as controls. It is convenient to asymmetrically position three patches of the positive control clone such that the signal on the autoradiograph may be used for orientation of the plate. Incubate at 30 degrees C overnight. Plates may be stored at 4 degrees C following incubation.

1. Prepare 82 mm round nylon filters by labeling with information identical to each patch plate. Autoclave the filters between pieces of 3MM Whatman.
2. Thoroughly warm the plates if they were stored at 4 degrees C. Lift the colonies from the surface of the plate by placing the corresponding filter face down onto the plate surface (handle the filter with filter forceps). Be careful to avoid bubbles beneath the filter. DO NOT lift the filter and replace.
3. Allow the filter to sit in place ~ 5 minutes. Then carefully peel the filter from the plate's surface and place it (cell side up) onto a fresh media plate, again avoiding bubbles beneath the filter. Incubate both sets of plates (original colonies and lifted colonies) at 30 degrees C overnight.

Day 3

1. Prepare SCE/ DTT/ Lyticase solution just before use. Cut one piece of 10 cm x 10 cm 3MM Whatman paper for each filter to be processed. Place the Whatman paper in a 150 mm petri dish. Soak the paper with 6 ml of the prepared SCE/ DTT/ Lyticase solution. Remove any bubbles by lifting the paper from one end and carefully replacing.
2. Lift the filter from the growth medium and place it (cell side up) on the saturated paper. There should be no bubbles beneath the filter.
3. Cover the membrane with the petri dish cover. Put several plates into a polyethylene bag and heat seal the bag. Incubate overnight at 30 degrees C (no longer than 24 hours).
4. Refrigerate the original plates. These plates may be lifted again if necessary.

Day 4

Cell lysis and DNA binding:

1. Line the bench with a fresh sheet of plastic wrap. Place10 cm x 10 cm pieces of Whatman on the wrap in positions suitable to the number of filters being processed. Use 6 ml of solution to soak each Whatman paper, lifting the paper from one end to avoid bubbles. Between different solution treatments lay out fresh plastic wrap and paper.
2. The filters are placed on the following solutions for the given times:
   For cell lysis:

   5 minutes 10% SDS

   10 minutes 0.5 N NaOH

   For neutralization and washing:

   5 minutes 200 mM Tris, pH7.5/2X SSC

   5 minutes " " "

   5 minutes " " "

3. Place the filters on a sheet of 3MM Whatman to air dry for at least 1 hour, then bake the filters for 1 hour in 80Å¡C vacuum oven, between pieces of Whatman. Store the filters dry, between the pieces of Whatman, or use immediately.

1. Place 10 -12 filters in a hybridization bag and wash twice by adding ~25 ml of sterile dH20, swishing through the bag and dumping the water. Add 10 ml of prehyb solution containing the necessary competitor DNA to the bag. Swish the prehyb solution throughout, then heat seal. Put the bag in a constant temperature incubator on a platform rocker.
2. Prepare the DNA probe by labeling with 32P following standard protocols. Add the radioactively labeled probe to 10 ml of fresh prehyb solution with competitor DNA.
3. Drain the bag of the old prehyb, and then add the fresh prehyb with the probe. Swish the the bag gently, heat seal, and swish again more vigorously. SHIELD ALL RADIOACTIVE WORK.
4. Place bag on a rocking platform in the appropriate constant temperature incubator overnight.

Day 5

1. Wash the filters by cutting one edge of the bag and pouring out the hybridization solution and unbound probe into the RAM waste.
2. Pour in ~25 ml of 1 mM Tris pH 7.5 / 1% Sarkosyl solution. Swish this around inside the bag and pour into RAM waste. Repeat this step 8-10 times.
3. Remove filters from bag and wash in 250 ml 1mM Tris pH 7.5 by shaking for 5 minutes at room temperature. Repeat this step 4 times.
4. Blot the filters dry on absorbent paper, but do not allow them to dry completely. Place the filters in a plastic exposure bag in an X-ray film holder containing an intensifying screen before setting up the exposure in the darkroom. Expose the film overnight at -80 degrees C.

Day 6

1. Thoroughly warm the film holder before developing the film. Label the film with the corresponding filter number.
2. Determine which clones hybridized to the DNA probe based on the film exposure. Compare the positive control hybridization to that of the clones being tested.

Begin the secondary screening procedure:

1. Using the original patch plate, touch the patch of a positive clone with an inoculating loop and streak out the cells on a fresh selective media plate. Incubate the plates for 3 days at 30 degrees C to obtain isolated colonies.

Day 9

1. Patch five isolated colonies for each positive clone onto selective media plates. Arrange the pattern such that five clones and the positive and negative controls will fit on each plate.Incubate at 30 degrees C overnight.

Day 10

1. Repeat steps as listed starting from Day 2 above.

Solutions:

• SCE solution:
  1M sorbitol

  100 mM sodium citrate

  60 mM EDTA

• SCE/ DTT/ Lyticase: (for 40 ml, prepare fresh each time):
  40 ml SCE

  300 µl 2 M DTT

  5600 units Lyticase

• 5X HPB (High Phosphate Buffer) (for 1 liter)
  146.1 g NaCl

  134.0 g Na2HPO4

  9.3 g Na2EDTA

  pH to 7.0 with concentrated HCl

  Autoclave to sterilize.

• 1mM Tris/1% Sarkosyl (for 1 liter)
  1 ml 1M Tris pH7.5

  100 ml 10% sarkosyl

  899 ml dH2O

• 200 mM Tris/2X SSC: (1 liter)
  200 ml 1M Tris pH 7.5

  100 ml 20 X SSC

  700 ml dH20

• Yeast prehyb/hyb solution: (for 10 ml)
  7 ml sterile dH2O

  2 ml 5X HPB

  1 ml 10% sarkosyl

References:

Brownstein, B. H., Silverman, G.A., Little, R.D., Burke, D. T., Korsmeyer, S. J., Schlessinger, D., and M. V. Olson, (1989) "Isolation of single-copy human genes from a library of Yeast Artificial Chromosome clones". Science 244: 1348-1351.

Feinberg, A.P. and B. Vogelstein. (1983). "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity" Anal. Biochem. 132: 6-13.