Generation Of A Subtracted cDna Library

In Vitro Packaging

Performance of packaging in vitro is to use a phage-infected E. coli cell extract to supply the mixture of proteins and precursors required for encapsulating the recombinant 1DNA (vector-cDNA inserts). Packaging systems are commercially available with the specific bacterial strain host and control DNA.

1. Thaw a packaging extract (50 ml/extract) on ice.

Note: Do not thaw the extract at room temperature or 37° C, and do not freeze the extract once it has thawed.

2. Once the extracts have thawed, immediately add 5 to 10 ml of the ligated mixture to the extract and mix gently.

3. Incubate at 22 to 24°C for 2 to 4 h and add 0.5 ml of phage buffer and 20 m1 of chloroform to the mixture. Gently mix well and allow the chloroform to settle to the bottom of the tube. Store the packaged phage at 4°C for up to 5 weeks, although the titer may drop. Proceed to titration of lambda phage.

Titration of Packaged Phage

1. Briefly thaw the specific bacterial strain such as E. coli DH5a, E. coli 392, Y 1090, Y1089 and KW 251 on ice. The bacterial strain is usually kept in 20% glycerol and stored at -70°C until use. Pick up a small amount of the bacteria using a sterile wire transfer loop and immediately inoculate the bacteria on the surface of an LB plate (Figure 3.5). Invert the inoculated LB plate and incubate it in an incubator at 37°C overnight. Multiple bacterial colonies will form.

Note: The LB plate should be freshly prepared and dried at room temperature for a couple of days prior to use.

2. Prepare fresh bacterial culture by inoculating a single colony from the streaked LB plate using a sterile wire transfer loop in 5 ml of LB medium supplemented with 50 ml of 20% maltose and 50 ml of 1 M MgSO4

FIGURE 3.5 Streaking of bacterial stock in a zig-zag pattern on an LB plate. Well-isolated bacterial colonies are generated 14 h later in the last streaking area in the plate.

solution. Shake at 160 rpm at 37°C for 6 to 9 h or until the OD600 has reached 0.6.

3. Dilute each of the packaged recombinant phage samples at 1000X, 5000X, 10,000X and 100,000X in phage buffer.

4. Add 20 ml of 1 M MgSO4 solution and 2.8 ml of melted top agar to 36 sterile glass tubes in a sterile laminar flow hood. Cap the tubes and immediately place them in 50°C water bath for at least 30 min prior to use.

5. Mix 0.1 ml of the diluted phage with 0.1 ml of freshly cultured bacterials in a microcentrifuge tube. Cap the tube and allow the phage to adsorb the bacteria in an incubator at 37°C for 30 min.

6. Add the phage-bacterial mixture into specified tubes in the water bath. Vortex gently and immediately pour onto the center of the LB plates, quickly overlaying the complete surface of the plate with the mixture by gently tilting the plate. Cover the plates and allow the top agar to harden for 10 min. Invert the plates and incubate them in an incubator at 37°C overnight or for 15 h.

Note: The top agar mixture should be evenly distributed over the surface of the LB plate. Otherwise, the growth of bacteriophage will be affected by decreasing the pfu number.

7. Count the number of plaques for each plate and calculate the titer of the phage (pfu).

Plaque forming units (pfu) per milliliter = number of plaques/plate x dilution times x 10

The last "10" of the calculation refers to the 0.1 ml, per milliliter basis, of the packaging extract used for one plate. For instance, if are 100 plaques are on a plate made from a 1/5000 dilution, the pfu per milliliter of the original packaging extract = 100 x 5000 x 10 = 5 x 106.

Reagents Needed

Ligase 10X Buffer

300 mM Tris-HCl, pH 7 8 0.1 M MgCl2 0.1 M DTT 10 mM ATP

Phage Buffer

0.1 M NaCl

10 mM MgSO4

LB (Luria-Bertaini) Medium (per Liter) 10 g Bacto-tryptone 5 g Bacto-yeast extract 5 g NaCl

Adjust to pH 7.5 with 0.2 N NaOH and autoclave. 20% (v/v) maltose 1 M MgSO4

LB Plates

Add 15 g of Bacto-agar to 1 l of prepared LB medium and autoclave. Allow the mixture to cool to about 60°C and pour 30 ml/dish into 85- or 100-mm Petri dishes in a sterile laminar flow hood with filtered air flowing. Remove any bubbles with a pipette tip and let the plates cool for 5 min prior to their being covered. Allow the agar to harden for 1 h and store the plates at room temperature for up to 10 days or at 4°C in a bag for 1 month. The cold plates should be placed at room temperature for 1 to 2 h before use.

TB Top Agar

Add 3.0 g agar to 500 ml prepared LB medium and autoclave. Store at 4°C until use. For plating, melt the agar in a microwave. When the solution has cooled to 60°C, add 0.1 ml of 1 M MgSO4 per 10 ml of the mixture. If color selection methodology is used to select recombinants in conjunction with bacterial host strain Y1090, 0.1 ml IPTG (20 mg/ml in water, filter-sterilized) and 0.1 ml X-Gal (50 mg/ml in dimethylformamide) should be added to the cooled (55°C) 10 ml of the top agar mixture.

S Buffer

Amplification of cDNA Library (Optional)

1. For each 50 ml of bacterial culture, add 10 to 15 ml of 1 x 105 pfu of packaged bacteriophage or total plaque eluate into a microcentrifuge tube containing 240 to 235 ml of phage buffer. Mix with 0.25 ml of cultured bacteria and allow the bacteria and phage to adhere to each other by incubating at 37°C for 30 min.

2. Add this mixture to a 250- or 500-ml sterile flask containing 50 ml of LB medium prewarmed at 37°C, which is supplemented with 1 ml of 1 M MgSO4. Incubate at 37°C with shaking at 260 rpm until lysis occurs.

Tips: It usually takes 9 to II h for lysis to occur. The medium should be cloudy after several h of culture and then be clear upon cell lysis. Cellular debris also becomes visible in the lysed culture. There is a density balance between bacteria and bacteriophage; if the bacteria density is much over that of bacteriophage, it takes longer for lysis to occur, or no lysis takes place at all. In contrast, if bacteriophage concentration is much over that of bacteria, lysis is too quick to be visible at the beginning of the incubation and, later on, no lysis will happen. The proper combination of bateria and bacteriaphage used in step I will assure success. In addition, careful observations should be made after 9 h of culture because the lysis is usually quite rapid after that time. Incubation of cultures should stop once lysis occurs; otherwise, the bacteria grow continuously and the cultures become cloudy again. Once that happens, it will take long time to see lysis, or no lysis will take place.

3. Immediately centrifuge at 9000 x g for 10 min at 4°C to spin down the cellular debris. Transfer the supernatant containing the amplified cDNA library in bacteriophage particles into a fresh tube. Aliquot the supernatant, add 20 to 40 ml of chloroform and store at 4°C for up to 5 weeks.

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