Subcloning of Yeast Artificial Chromosomes into Phage Lambda

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 礸 of DNA, 2 祃 of 10X ligation buffer, 2 祃 of 10 mM ATP pH 7.5, 8 units of T4 DNA ligase (keep glycerol content below 5%), and add ddH2O to a 20 祃 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 祃 volume (use 5 祃 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 礸 of DNA to tube 1, 15 礸 to tubes 2-4 and 6, and no DNA to tube 5.

2. Add 15 祃 of 10X MboI restriction buffer to tubes 2-4 and 6, 30 祃 to tube 1, and none to tube 5. The contents of each tube, at this point in the procedure, are summarized below:

   Total Volume (祃)	DNA (礸)	10X Buffer (祃)
   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 祃 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 祃 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 (祃)	DNA (礸)	10X Buffer (祃)	MboI units/15礸DNA 上一篇：Identification of End Clones in YAC Subclone Libraries   下一篇：Sizing of YAC Clones 共3页: 上一页 1 [2] [3] 下一页