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May 5, 1990
Jim Howe
Purpose:
Time required:
Special Reagents:
Procedure:
Vector Preparation
Day 1
Day 2
Insert Preparation
Days 1-5
Day 6
Prepare an MboI partial digestion of the yeast liquid DNA by the enzyme dilution method:
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 |
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 | ----- |
Day 7
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
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
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:
Days 9-10
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.
Solutions:
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.