II. Random subclone generation
A. Sonication
The generation of DNA fragments by sonication is performed by placing a microcentrifuge tube containing the buffered DNA sample into an ice-water bath in a cup-horn sonicator and sonicating for a varying number of 10 second bursts using maximum output and continuous power (10), essentially as described by Bankier and Barrell (11). During sonication, temperature increases result in uneven fragment distribution patterns, and for that reason, the temperature of the bath is monitored carefully during sonication, and fresh ice-water is added when necessary. The exact conditions for sonication are determined for a given DNA sample before a preparative sonication is performed. Approximately 100 ug of DNA sample, in 350 ul of buffer, is distributed into ten aliquots of 35 ul, five of which are subjected to sonication for increasing numbers of 10 second bursts. Aliquots from each time point are electrophoresed on an agarose gel versus the phi-X 174 size marker (12) to determine the approximate DNA fragment size range for each sonication time point. Once optimal sonication conditions are determined, the remaining five DNA aliquots (approximately 50 ug) are sonicated according to those pre-determined conditions. After sonication, the five tubes are placed in an ice-water bath until fragment end-repair and size selection, discussed below.
Protocol
1. Prepare the following DNA dilution, and aliquot 35 ul into ten 1.5 ml microcentrifuge tubes:
DNA 100 ug
10X TM buffer 35 ul
sterile ddH2O q.s.
Final Volume 350 ul
2. To determine the optimal sonication conditions, sonicate the DNA samples in five of the tubes in a Heat Systems Ultrasonics W-375 cup horn sonicator set on 'HOLD', 'CONTINUOUS', and maximum 'OUTPUT CONTROL' = 10 under the following conditions:
Tube No. 10 second bursts
1 1
2 2
3 3
4 4
5 5
We have recently learned that the Genome Center at Washington University and the Sanger Center set the OUTPUT CONTROL to the lowest possible settings. Because at present we use the Nebulizer (see the next section below), we have not investigated this further.
2. Cool the DNA samples by placing the tubes in an ice-water bath for at least 1 minute between each 10 second burst. Replace the ice-water bath in the cup horn sonicator between each sample.
3. Centrifuge the samples to reclaim condensation and electrophorese a 10 ul aliquot from each sonicated DNA sample on a agarose gel versus the phi-X 174/HaeIII size marker (Pharmacia 15611-015).
4. Based on the fragment size ranges detected from agarose gel electrophoresis, sonicate the remaining 5 tubes according to the optimal conditions and then place the tubes in a ice-water bath.
B. Nebulization
You can purchase Nebulizer, Number 4101 or 4101UO, from a local supplier, whose name you can obtain by calling the manufacturer:
IPI Medical Products Inc.
3217 North Kilpatrick
Chicago, IL 60641
phone: (773) 777-0900
The president of IPI is Walter Levine so if you have any troubles ordering them be sure to ask for him and/or to tell them that these devices are:
"NOT INTENDED FOR PATIENT USE"
Basically we follow a protocol sent to us by Steve Surzycki at the Department of Biology, Indiana University.
There are two small problems that we solved as follows:
1. You have to cover the hole where normally the mouth piece gets attached to; cover that hole with a cap QS-T from ISOLAB Inc. (Drawer 4350 Akron, OH 44303, 100 caps for $ 9.50).
2. The other problem that may occur is that the nebulizer leaks where the hose for the nitrogen gets attached. It seems that Nalgene tubing (VI grade 3/16" ID) seals better that the tubing which comes with the nebulizer. The nebulizer might still leak somewhat at the top, you can't avoid that.
Nebulizer Summary:
A nebulizer containing 2 ml of a buffered DNA solution (approximately 50 ug) containing 25-50% glycerol is placed in an ice-water bath and subjected to nitrogen gas at a pressure of 8-10 psi for 2.5 minutes for nebulizing BACs (10,13). Nitrogen gas pressure is the primary determinant of DNA fragment size, and although pressure studies should be performed with each BAC, cosmid or plasmid, a pressure of 8-10 psi almost always resulted in the desired (1kb-4kbp) fragment size range. As discussed above for sonication, the use of an ice-water bath for nebulization also is critical to the generation of evenly distributed DNA fragments. During the nebulization process, unavoidable leaks are minimized by securely tightening the lid for nebulizer chamber and sealing the larger hole in the
top piece with a plastic cap. To prepare for fragment end-repair, the nebulized DNA typically is divided into four tubes and concentrated by ethanol precipitation.
Protocol
1. Modify a nebulizer (IPI Medical Products, Inc. 4207) by removing the plastic cylinder drip ring, cutting off the outer rim of the cylinder, inverting it and placing it back into the nebulizer. Seal the large hole inthe top cover (where the mouth piece was attached) with a plastic stopper and connect a 1/4 inch id length of Tygon tubing (which eventually should beconnected to a compressed air source) to the smaller hole.
2. Prepare the following DNA sample and place in the nebulizer cup:
DNA 50 ug
10X TM buffer 200 ul
sterile glycerol 0.5-1 ml
sterile ddH2O q.s.
2 ml
3. Nebulize in an ice-water bath at 30 psi for 2.5 minutes for plasmid, or 8-10 psi for 2.5 minutes for BACs, PACs, fosmids or cosmids.
4. Briefly centrifuge at 2500 rpm to collect the sample by placing the entire unit in the rotor bucket of a table top centrifuge (Beckman GPR tabletop centrifuge) fitted with pieces of styrofoam to cushion the plastic nebulizer.
5. Distribute the sample into four 1.5 ml microcentrifuge tubes and ethanol precipitate. Resuspend the dried DNA pellet in 35 ul of 1X TM buffer prior to proceeding with fragment end-repair.
C. Random fragment end-repair, size selection, and phosphorylation
Since both sonicated and nebulized DNA fragments usually contain single-stranded ends, the samples are end-repaired prior to ligation into blunt-ended vectors (10,11). A combination of T4 DNA polymerase and Klenow DNA polymerase are used to "fill-in" the DNA fragments by catalyzing the 3'-5' incorporation of complementary nucleotides into resultant double-stranded fragments with a 5' overhang. Additionally, the single-stranded 3'-5' exonuclease activity of T4 DNA polymerase is used to degrade 3' overhangs. The reactions included the two enzymes, buffer, and deoxynucleotides and are incubated at 37degC.
Following fragment end-repair, the DNA samples are electrophoresed on a preparative low-melting temperature agarose gel versus the phi-X 174 marker, and after appropriate separation, the fragments in the size range from 1-2Kbp and 2-4Kbp are excised and eluted separately from the gel, as discussed above. Alternatively, the fragments can be purified by fractionation on a Sephacryl S-500 spin column as also discussed above. In both instances, the purified fragments are concentrated by ethanol precipitation followed by resuspension in kinase buffer, and phosphorylation using T4 polynucleotide kinase and rATP. The polynucleotide kinase is removed by phenol extraction and the DNA fragments are concentrated by ethanol precipitation, dried, resuspended in buffer, and ligated into blunt-ended cloning vectors. It should be noted that because a significant portion of nebulized DNA fragments are easily cloned without end-repair or kinase treatment, these two steps can be combined without significantly affecting the overall number of resulting transformed clones (see section V.B. on purification of PCR fragments for cloning, which describes a method for simultaneous end-repair and kinase treatment).
Protocol
1. To each tube containing 35 ul of DNA fragments (five of sonicated DNA and four of nebulized DNA), add:
0.25 mM dNTPs 2 ul
T4 DNA polymerase 3 ul (3 U/ul)
Klenow DNA polymerase 2 ul (5 U/ul)
42 ul
T4 (203L) and Klenow (210L) DNA polymerases from New England Biolabs.
2. Incubate at room temperature for 30 minutes.
3a. Add 5 ul of agarose gel loading dye and apply to separate well of a 1% low gel temperature agarose gel and electrophorese for 30-60 minutes at 100-120 mA.
4a. Elute the DNA from each sample lane, ethanol precipitate, and resuspend the dried DNA in 36 ul of sterile ddH2O and add 4 ul of 10X denaturing buffer. There should be five tubes for sonicated fragments and four tubes for nebulized fragments.
5a. Incubate at 70degC for 10 minutes, and place the samples in an ice-water bath.
6a. Add the following reagents for the kinase reaction and incubate at 37 degC for 10-30 minutes:
10 mM rATP 1 ul
10 X kinase buffer 5 ul
T4 polynucleotide kinase 1 ul (30 U/ul)
Final Volume 47 ul
T4 polynucleotide kinase (70031) from United States Biochemicals.
7a. Pool the kinase reactions, phenol extract, ethanol precipitate, and resuspend the dried DNA fragments in 40 ul of 10:0.1 TE buffer. This yields a typical concentration of 500-1000 ng/ul.
Alternatively the end-repair and phosphorylation steps can be combined:
1b. Resuspend DNA in 27 ul of 1X TM buffer. Add the following:
10X kinase buffer 5 ul
10 mM rATP 5 ul
0.25 mM dNTPs 7 ul
T4 polynucleotide kinase 1 ul (3 U/ul)
Klenow DNA polymerase 2 ul (5 U/ul)
T4 DNA polymerase 3 ul (3 U/ul)
------------------------------------------------------
Final Volume