Method: Subcloning of Yeast Artificial Chromosomes into Phage Lambda

May 5, 1990

Jim Howe

Purpose:

To subclone the large insert fragment of human DNA contained within a YAC into a bacteriophage lambda vector. The subclones are 15 to 23 kb in size, and can be used to identify new polymorphic markers from a known region of the genome, to map a specific locus, and/or to screen other libraries.

Time required:

10-12 days

Special Reagents:

• Restriction enzyme MboI
• EMBL3 or other phage vector
• Packaging Extract (Stratagene Gigapack II XL)

Procedure:

Vector Preparation

Day 1

1. Ligate the cos sites of the lambda vector (we have used EMBL3 from Stratagene) as follows. Combine 10 µg of DNA, 2 µl of 10X ligation buffer, 2 µl of 10 mM ATP pH 7.5, 8 units of T4 DNA ligase (keep glycerol content below 5%), and add ddH2O to a 20 µl final volume.
2. Incubate the reaction mix for 24 hours at 4 degrees C. Add 8 additional units T4 DNA ligase and incubate 24 hours more.
3. Heat inactivate ligase at 68 degrees C for 15 minutes.

1. Digest with 50 units BamHI and 50 units of EcoRI at 37 degrees C for 2 hours in a 50 µl volume (use 5 µl of Stratagene 10X Universal Buffer). Run an aliquot out on a gel to ensure complete digestion. BamHI digested DNA ends have the same overhang as MboI, and it is here that the human inserts will be ligated. EcoRI inactivates the 13 kb phage stuffer fragment by eliminating the complementary BamHI overhangs (EcoRI sites are just within these Bam sites)
2. Phenol/chloroform extract the vector DNA. Follow with an ethanol precipitation, wash the precipitate in 70% ethanol, then resuspend the DNA pellet in TE. Run an aliquot out on a gel to determine the vector DNA concentration.

Days 1-5

1. Prepare high molecular weight liquid DNA. (Protocols for DNA preparation are given in the Methods section for restriction enzyme digestions.)

Day 6

Prepare an MboI partial digestion of the yeast liquid DNA by the enzyme dilution method:

1. Label 5 eppendorf tubes 1-6, then add 30 µg of DNA to tube 1, 15 µg to tubes 2-4 and 6, and no DNA to tube 5.
2. Add 15 µl of 10X MboI restriction buffer to tubes 2-4 and 6, 30 µl to tube 1, and none to tube 5. The contents of each tube, at this point in the procedure, are summarized below:

   Total Volume (µl)	DNA (µg)	10X Buffer (µl)
   1. 300	30	30
   2. 150	15	15
   3. 150	15	15
   4. 150	15	15
   5. ---	--	--
   6. 150	15	15

3. Add 2 units of MboI (dilute enzyme so that this amount can be delivered accurately) to tube 1, cap, and mix gently to achieve good distribution of enzyme; be gentle to avoid shearing of DNA.
4. Pipette 150 µl of the mixture from tube 1(using pipette tips with their ends cut off and sterilized) into tube 2, cap, mix, and repeat this down the line of tubes until tube 5. After 150 µl has been added to tube 5, add 2 units of MboI and mix gently. Do not add enzyme to tube 6, for this will serve as a control to show the quality of the DNA. The table below summarizes the contents of each tube at this point in the procedure:

   Total Volume (µl)	DNA (µg)	10X Buffer (µl)	MboI units/15µgDNA
   1. 150	15	15	1.0
   2. 150	15	15	0.5
   3. 150	15	15	0.25
   4. 150	15	15	0.125
   5. 150	15	15	2.0
   6. 150	15	15	-----

5. Add 1-2 µl of RNase (5 mg/ml) to each tube, then place these tubes at 37 degrees C for exactly one hour. Heat to 70 degrees C for 15 minutes to inactivate the restriction enzyme.
6. Load a 15 µl aliquot from each tube onto a 0.5% agarose gel (load in order 5,1,2,3,4,6); load standards in adjacent lanes to include uncut lambda, lambda pre-digested with BglII or HindIII, lambda cut with XhoI, and 1 kb ladder. Run the gel at 60V for 14 hours using a pump to recirculate the buffer.

Day 7

1. Stain the gel with ethidium bromide and photograph. Examine the gel photograph and determine which dilutions give the greatest proportion of the total smear in the 15-23 kb range. This will be the ideal dilution for subcloning because these are optimal insert sizes for the EMBL3 phage vector.
2. To the selected digests, add 20 µl of Calf Intestinal Alkaline Phosphatase (CIP) dephosphorylation buffer (10 mM ZnCl2, 10 mM MgCl2, 100 mM Tris-HCl pH 8.3), and 27 µl dH2O. Add 2-3 units of CIP and put the tubes in a 50 degrees C heat block for 30 minutes. Add 2-3 more units, then incubate an additional 30 minutes.
3. Heat to 70 degrees C for 15 minutes to inactivate the CIP.

   One now has two options (a or b) for subcloning: option (a) takes one day less than option (b) and is more likely to result in successful ligation. Option (b) is more specific for generating clones with the desired size inserts:

   (a) subclone from the tube of DNA after phenol /chloroform extraction, reprecipitation with ethanol and determining the concentration of fragments; there will be some fragments larger and smaller than the ideal size range using this method, but the use of size-selctive packaging mix (Gigagpack II XL, Stratagene) will minimize the effect of this.

   (b) one can run the entire tube out in multiple lanes of a 0.5% agarose gel as before, and now cut out the fragments within the desired size range; one must then recover the DNA by electroelution, phenol/chloroform extraction, reprecipitation with ethanol, and then determine the concentration of the DNA prior to ligation.

Ligation and Packaging

Day 8-9

1. Set up ligation reaction as follows:
   1.0 µg EMBL3 digested arms

   0.4 µg Insert DNA

   1.0 µl of 10X Ligase buffer

   1.0 µl 10 mM ATP

   1.0 µl T4 DNA Ligase (2-4 Weiss units)

   Adjust volume to 10 µl with dH2O

2. Place the reaction mix at room temperature for 30-60 minutes, then incubate at 4 degrees C for 24-48 hours. Run a small aliquot out on a mini-gel to determine whether the ligation was successful.

   Follow the DNA packaging protocol appropriate for the packaging kit you will be using. We have used the Stratagene Gigapack II XL kit which is useful for both phage and cosmid packaging, and packages 20 kb inserts with 95% greater efficiency than 14 kb inserts. To package the DNA, do the following:

3. Remove the freeze/thaw extract and the sonic extract from the -80 degrees C freezer and place on ice.
4. Quickly thaw the freeze/thaw extract by holding the tube between the fingers until it just begins to thaw.
5. Immediately add 4 µl of the ligation reaction to the freeze/thaw extract and place on ice.
6. Thaw the sonic extract and add 15 µl to the freeze/thaw tube, then incubate at 22 degrees C for 2 hours. Add 500 µl of SM buffer (see phage solutions), 20 µl of chloroform and mix gently. Centrifuge to sediment debris. The packaging mixture can be stored indefinitely at 4 degrees C.

Plating

Days 9-10

1. Start a 25 ml overnight bacterial culture with strain P2392.
2. At this point, one has intact phage particles in this packaging mix, which represent the phage library of this YAC. The phage need to be titered by making several dilutions and adsorbing to 200 µl of bacterial strain P2392 for 15 minutes, adding to 3 ml top agar, then plating on LB plates and incubating 12-18 hours.
3. Select the dilution that gives the largest number of plaques which can be separated cleanly from one another. This is usually around 200 to 400 plaques per 82 mm plate. To obtain clones spanning the entire YAC clone, it is desirable to have a 3 to 5 fold coverage of the yeast genome; for smaller YACs, the even higher coverage may be needed. To determine how many of these plates are needed in order to have a 3 to 5 hit coverage of the YAC, do the following calculations:

   Size of Yeast Genome:	1.5 X 107 bp
   Size of average YAC:	3.0 X 105 bp
   Size of average Inserts:	2.0 X104 bp
   # Inserts to cover Genome:	750
   Number of plaques to screen to cover
   the YAC genome 3-5 times:	2250-3750
   Number of plates needed (375 plaques each):	10

   It is important to remember that you have cloned the entire yeast genome as well as the YAC into the phage, and thus need to devise a strategy of recovering human containing recombinants.

4. Prepare filters from each plate (as described in phage methods section for plaque lifts), and hybridize with total human DNA to identify the human clones by the presence of highly repetitive sequences, e. g., Alu sequences. Hybridization of the plaque lifts to total yeast DNA identifies yeast clones with yeast repetitive DNA. Using probes you know are located on the YAC to screen the YAC subclones will identify clones containing those sequences. The ends of the human insert can be identified by hybridizing the plaque lifts to pBR322 to detect the subclones containing portions of the vector arms (see the procedure for the identification of end clones in YAC subclone libraries).

Solutions:

• Mbo I Buffer:
  100 mM NaCl

  10 mM Tris-HCl, pH 7.4

  10 mM MgCl2

  1 mM Dithiothreitol

  100 µg/ml bovine serum albumin

References:

Sambrook, J., Fritsch, E.F., and T. Maniatis.(1989) Molecular Cloning, A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press. p. 2.32, 2.82-2.98.

Instruction manual to Stratagene Undigested EMBL3 Cloning Kit, 1989.

Instruction manual to Gigapack II XL Packaging Extract protocol, 1989.

---