Screening YAC libraries

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 80欳 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
   

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