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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.

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                                 50 ul              note: if the DNA has been sheared by nebulizing,                   the T4 DNA polymerase addition here may not                   be necessary.  
2b. Incubate at 37degC for 30 minutes

3b. Add 5 ul of agarose gel loading dye and apply to separate well of a 1% low melting temperature agarose gel and electrophorese for 30-60 minutes at 100-120 mA.

4b. Elute the DNA from each sample lane, ethanol precipitate, resuspend in 10 ul of 10:0.1 TE buffer.

D. DNA ligation

DNA ligations are performed by incubating DNA fragments with appropriately linearized cloning vector in the presence of buffer, rATP, and T4 DNA ligase (10,11). For random shotgun cloning, sonicated or nebulized fragments are ligated to either SmaI linearized, dephosphorylated double-stranded M13 replicative form or pUC vector by incubation at 4degC overnight. A practical range of concentrations is determined based on the amount of initial DNA, and several different ligations, each with an amount of insert DNA within that range, are used to determine the appropriate insert to vector ratio for the ligation reaction. In addition, several control ligations are performed to test the efficiency of the blunt-ending process, the ligation reaction, and the quality of the vector (10,11). These usually included parallel ligations in the absence of insert DNA to determine the background clones arising from self-ligation of inefficiently phosphatased vector. Parallel ligations also are performed with a known blunt-ended insert or insert library, typically an AluI digest of a cosmid, to insure that the blunt-ended ligation reaction would yield sufficient insert containing clones, independent of the repair process.

Protocol

1. Combine the following reagents in a microcentrifuge tube, and incubate overnight at 4degC:

 		DNA fragments		  100-1000 ng 		cloning vector				 2 ul (10 ng/ul) 		10X ligation buffer	 		 1 ul 		T4 DNA ligase (NEB 202L) 	 1 ul (400 U/ul) 		sterile ddH2O			  q.s. 								10 ul 
The cloning vector typically is SmaI-linearized, CIAP-dephosphorylated pUC vector (Pharmacia 27-4860-01) as several years ago we switched from M13 to pUC-based shotgun cloning. The advantage of obtaining two sequence reads off one isolated shotgun sub-clone seems to outweigh the disadvantage of a few bases less in double-stranded vs single-stranded read lengths. In some instances, including 5% PEG in the ligation reactions also seems to slightly improve the ligation efficiency.

2. Include control ligation reactions with no insert DNA and with a known blunt-ended insert (such as AluI digested cosmid).

E. Competent cell preparation

There are two main methods for preparation of competent bacterial cells (14) for transformation, the calcium chloride and the electroporation method. For the calcium chloride method, a glycerol cell culture stock of the respective E. coli strain is thawed and added to 50 ml of liquid media. This culture then is preincubated at 37degC for 1 hour, transferred to an incubator-shaker, and is incubated further for 2-3 hours. The cells are pelleted by centrifugation, resuspended in calcium chloride solution, and incubated in an ice-water bath. After another centrifugation step, the resulting cell pellet again is resuspended in calcium chloride to yield the final competent cell suspension. Competent cells are stored at 4degC, for up to several days.

Calcium Chloride Protocol

1. Thaw a frozen glycerol stock of the appropriate strain of E. coli, add it to an Erlenmeyer flask containing 50 ml of pre-warmed 2xTY (1) media, and pre-incubate in a 37degC water bath for 1 hour with no shaking. Further incubate for 2-3 hours at 37degC with shaking at 250 rpm.

2. Transfer 40 ml of the cells to a sterile 50 ml polypropylene centrifuge tube, and collect the cells by centrifugation at 3000 rpm for 8 minutes at 4deg C in a GPR centrifuge (Beckman) or 6000 rpm for 8 minutes at 4degC in an RC5-B centrifuge (DuPont) equipped with an SS-34 rotor. For M13-based transformation, save the remaining 10 ml of culture in an ice-water bath for later use.

3. After centrifugation, decant the supernatant and resuspend the cell pellet in one-half volume (20 ml) of cold, sterile 50 mM calcium chloride, incubate in an ice-water bath for 20 minutes, and centrifuge as before.

4. Decant the supernatant and gently resuspend the cell pellet in one-tenth volume (4 ml) of cold, sterile 50 mM calcium chloride to yield the final competent cell suspension.

Preparation of calcium chloride competent cells for frozen storage

1. Transfer 166 ul of the competent cell suspension to sterile Falcon culture tubes.

2. Add 34 ul of sterile 100% glycerol to the 166 ul aliquots of the final competent cell suspension prepared above, giving a final concentration of 17 % glycerol.

3. The competent cells then should be placed at -70degC and can be stored indefinately.

4. To use competent cells for transformation, remove from freezer and thaw for a few minutes at 37degC. Place on ice, add plasmid DNA and incubate for one hour as in the standard transformation procedure. Then heat shock at 42degC for 2 minutes, cool briefly, add 1 ml of 2xTY and incubate for 1 hour at 37degC before spreading on plates.

Electroporation Protocol

Preparation of Electro-competent Cells:

1. Grow XL1-Blue cells on a tetracycline plate (20 ug tet/ml of LB agar)

2. Inoculate 3 ml of YENB and grow overnight at 37 degrees C with shaking at 250 rpm in the New Brunswick incubator shaker.

3. Inoculate the 3 ml of overnight growth into 1 liter of YENB (7.5 grams of Bacto Yeast Extract and 8 grams of Bacto Nutrient Broth brought to 1 liter with distilled water and autoclaved) and grow to an A600 of 0.5 (typically requires 3-4 hours of shaking at 250 rpm in the New Brunswick incubator shaker at 37 degrees C.

4. Distribute the 1 liter of cells into four 500 ml Sorval (GS-3) centrifuge bottles and centrifuge at 5000 rpm at 4 degrees C for 10 minutes.

Note: Steps 5-9 should be performed in the cold room and typically ~600 ml of ice cold sterile water and 150 ml of ice cold sterile 10% glycerol are required for manipulating the cells from a 1 liter growth.

5. Resuspend each pellet in 100 ml of ice cold sterile double distilled water and combine the resuspended pellets into two Sorval centrifuge bottles (i.e each bottle then will contain 200 ml of resuspended pellet).

6. Centrifuge at 5000 rpm at 4 degrees C for 10 minutes in the Sorval GS-3 Rotor.

7. Resuspend each of the two pellets in 100 ml of ice cold sterile double distilled water and combine the resuspended pellets into one Sorval centrifuge bottle and centrifuge at 5000 rpm at 4 degrees C for 10 minutes in the Sorval GS-3 Rotor once more. Note: The purpose of all these centrifugation/resuspension/centrifugation steps is to insure that the cells are essentially "salt-free" as salt causes arching during the electroporation step.

8. Resuspend the pellet in 100 ml of 10% ice cold sterile glycerol, centrifuge as above, and finally resuspend the pellet in 2 ml of 10% ice cold sterile glycerol to give salt-free, concentrated electrocompetent cells.

9. Aliquote 40 ul of these electrocompetent cells into small snap cap tubes and immediately freeze by placing in curshed dry ice and then store at -70 degrees C until needed.

Electroporation Protocol for transformations using double-stranded plasmids

1. Thaw the electro-competent cells on ice for about one minute.

2. Add 2-3 ul of the ligation mix to the cells.

3. transfer 40 ul of the cells into to BTX Electroporation cuvettes PLUS and MAKE SURE THAT THE CELLS COVER THE BOTTOM OF THE CUVETTE.

4. Turn on the Bio Rad E. coli Pulser and set the current to 2.5 KV by pushing the "Lower" and "Raise" bottoms simultaneously twice.

5. Place the cuvette in the holder and slide it into position.

6. Charge by pressing the "Charge" bottom until you hear the beep.

7. Immediately, suspend the cells in 1 ml of YENB and transfer into a Falcon tube.

8. Incubate the cells at 37 degrees C for 30 minutes at 250 rpm shaker.

9. Spin the cells in BECKMAN table-top centrifuge for 8 minutes at 2500 rpm

10. Resuspend the cells in 200 ul fresh YENB and add 30 ul of 20 mg/ml XGAL and 30 ul of 25 mg/ml IPTG

11. Plate ~130 ul of the cells on pre-warmed LB-amp plates.

Reference:
Rakesh C. Sharma and Robert T. Schimke, "Preparation of Electro-competent E. coli Using Salt-free Growth Medium", Biotechniques 20, 42-44 (1996).

F. Calcium Chloride treated bacterial cell transformation

A brief background discussion of transformation and transfection can be found in the Appendix.

For DNA transformation (14,15), the entire DNA ligation reaction is added to an aliquot of competent cells, which is mixed gently, and incubated in an ice-water bath. This mixture then is heat-shocked briefly in a 42degC water bath for 2-5 minutes. At this point in the transformation, the method varied slightly depending on whether the cloning vector is M13-based or pUC-based.

For M13-based transformation (14), an aliquot of non-competent cells is added to the heat-shocked mixture, as is the lac operon inducer homologue, IPTG, and the b-galactosidase chromogenic substrate, x-gal. Melted top agar is added, and the transformation mixture then is poured onto the surface of an agar plate. After the top agar solidified, the plates are inverted and incubated overnight at 37degC.

For pUC-based transformation (15), an aliquot of liquid media is added to the heat-shocked mixture, which then is incubated in a 37degC water bath for 15-20 minutes. After recovery, the cell suspension is concentrated by centrifugation and then gently resuspended in a smaller volume of fresh liquid media. IPTG and x-gal are added to the cell mixture, which is spread onto the surface of an ampicillin-containing agar plate. After the cell mixture had diffused into the agar medium, the plates are inverted and incubated overnight at 37degC.

Protocol

1. Add the entire ligation reaction to a 12 X 75 Falcon tube containing 0.2-0.3 ml of competent cells, mix gently, and incubate in an ice-water bath for 40-60 minutes. (For retransformation of recombinant DNA, add approximately 10-100 ng of DNA directly to competent cells).

2. Heat shock the cells by incubation at 42degC for 2-5 minutes.

For M13-based transformation:

3a. Add the following reagents to the heat shocked transformation mixture:

 		Non-competent cells		0.2 ml 		IPTG (25 mg/ml H2O)		 25 ul 		x-gal (20 ml/ml DMF)	 25 ul 		lambda top agar			2.5 ml 
4a. Mix by briefly vortexing, and then quickly pour onto the surface of a pre-warmed lambda agar plate.

5a. Allow 10-20 minutes for the agar to harden, and then invert and incubate overnight at 37degC.

For pUC-based transformation:

3b. Add the following reagents to the heat shocked transformation mixture, add 1 ml of fresh 2xTY and incubate in a 37degC water bath for 15-30 minutes.

4b. Collect the cells by centrifugation at 3000 rpm for 5 minutes, decant the supernatant, and gently resuspend in 0.2 ml of fresh 2xTY.

5b. Add 25 ul IPTG (25 mg/ml water) and 25 ul x-gal (20 mg/ml DMF), mix and pour onto the surface of a pre-warmed LB-Amp plate. Spread over the agar surface using a sterile bent glass rod or sterile inoculating loop.

6b. Allow 10-20 minutes for the liquid to diffuse into the agar, and then invert and incubate overnight at 37degC.

For pBR322, pAT153 or other non-lacZ containing vectors:

3b. Add 1 ml of fresh 2xTY to the cells and incubate for 15-30 minutes at 37 degC. Spread approximately 50 ul on L plates containing antibiotic using a sterile glass spreader. Incubate the plates overnight at 37degC.

G. Microcentrifuge Tube Transformation

Microcentrifuge transformations are recommended when a single plasmid is being retransformed or for qualitative transformation experiments. Shotgun cloning experiments should be transformed using the large scale transformation, since the objective is to efficiently obtain transformation of hundreds of distinct recombinant plasmids.

1. Inoculate 50 ml of fresh 2xTY media with 3 to 5 ml of a fresh overnight culture of a suitable host strain (GM272) and incubate for 2 to 3 hours at 37deg C.

2. Transfer 1 ml of the culture into a 1.5 ml tube and centrifuge for 5 min at room temperature. Use 1 tube of culture per DNA sample to be transformed.

3. Decant supernatant, and resuspend the cell pellet in 500 ul (1/2 volume) of sterile, cold 50 mM calcium chloride. Gently vortex if necessary.

4. Incubate 5 min. on ice.

5. Centrifuge as before, decant and resuspend the competent cell pellet in 100 ul (1/10 volume) of calcium chloride.

6. Transfer each 100 ul sample of competent cells to chilled 12 x 75 mm Falcon tubes which contain 3 to 5 ul of DNA sample (about 2 ng/ul to 20 ng/ul).

7. Incubate on ice for 15 minutes.

8. Heat shock the sample at 42degC for 5 minutes.

9. Add 1 ml of fresh 2xTY to each sample and recover the cells by incubating at 37degC for 15 min.

10. For lacZ containing vectors add 25 ul of 20 mg/ml IPTG (in water) and 25 ul of 24 mg/ml X-Gal (in DMF).

11. Add 2.5 ml of soft top agar to each sample, vortex and quickly pour onto the surface of a TYE-AMP agar plate. Allow at least 15-30 min. for the agar to solidify.

12. Invert the plates and incubate overnight at 37degC.


Bruce A. Roe, Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma 73019 broe@ou.edu