H. Fragment purification on Sephacryl S-500 spin columns

DNA fragments larger than a few hundred base pairs can be separated from smaller fragments by chromatography on a size exclusion column such as Sephacryl S-500. To simplify this procedure, the following mini-spin column method has been developed.

1. Thoroughly mix a fresh, new bottle of Sephacryl S-500, distribute in 10 ml portions, and store in screw cap bottles or centrifuge tubes in the cold room.

2. Prior to use, briefly vortex the matrix and without allowing to settle, add 500 ul of this slurry to a mini-spin column (Millipore) which has been inserted into a 1.5 ml microcentrifuge tube.

3. Following centrifugation at 2K RPM in a table top centrifuge, carefully add 200 ul of 100 mM Tris-HCl (pH 8.0) to the top of the Sephacryl matrix and centrifuge for 2 min. at 2K RPM. Repeat this step twice more. Place the Sephacryl matrix-containing spin column in a new microcentrifuge tube.

4. Then, carefully add 40 ul of nebulized cosmid, plasmid or P1 DNA which has been end repaired to the Sephacryl matrix (saving 2 ul for later agarose gel analysis) and centrifuge at 2K RPM for 5 minutes. Remove the column, save the solution containing the eluted, large DNA fragments (fraction 1). Apply 40 ul of 1xTM buffer and recentrifuge for 2 minutes at 2K RPM to obtain fraction 2 and repeat this 1xTM rinse step twice more to obtain fractions 3 and 4.

5. To check the DNA fragment sizes, load 3-5 ul of each eluant fraction onto a 0.7% agarose gel that includes as controls, 1-2 ul of a PhiX174-HaeIII digest and 2 ul of unfractionated, nebulized DNA saved from step 4 above.

6. The fractions containing the nebulized DNA in the desired size ranges (typically fractions 1 and 2) are separately phenol extracted and concentrated by ethanol precipitation prior to the kinase reaction.

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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.