Random subclone generation

(adapted fromBruce A. Roe, Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma 73019 broe@ou.edu)

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 μl of buffer, is distributed into ten aliquots of 35 μl, 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 μl into ten 1.5 ml microcentrifuge tubes:

DNA 100 ug 10X TM buffer 35 μl sterile ddH2O q.s. Final Volume 350 μl

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 μl 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 4207, from a local supplier, whose name you can obtain by calling the manufacturer:

IPI Medical Products Inc. 3217 North Kilpatrick Chicago, IL 60641 phone: (312) 777-0900

We basically 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.