Screening of the expression library and isolation of THE cDNA of interest

Method A. Immunoscreening of Expression cDNA Library Using Specific Antibodies cDNAs cloned at the multiple cloning site (MCS) in the lac Z gene of vector Igtll Sfi-Not I can be expressed as a part of a ß-galactosidase fusion protein. The expression cDNA library can be screened with specific antibodies of interest. The primary positive clones are then "fished" out from the library. Following several rounds of retitration and rescreening of the primary positive clones, putative cDNA clones of interest will be isolated.

1. Prepare LB medium, pH 7.5, by adding 15 g of Bacto-agar to 1 l of the LB medium followed by autoclaving. Allow the mixture to cool to about 55°C and add ampicillin (100 mg/ml) or tetracycline (15 mg/ml), depending on the antibiotic-resistant gene contained in the vectors. Mix well and pour 30 ml/dish of the mixture 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 allow the plates to cool for 5 min prior to being covered. Let the agar 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 prior to use.

2. Partially thaw a specific bacterial strain such as E. coli Y 1090 on ice. Pick up a tiny bit of the bacteria using a sterile wire transfer loop and immediately streak out E. coli Y1090 on a LB plate by gently drawing several lines on the surface of an LB plate (Figure 3.5). Invert the inoculated LB plate and incubate in an incubator at 37°C overnight. Multiple bacterial colonies will be generated.

3. Prepare fresh bacterial cultures by picking up a single colony from the streaked LB plate using a sterile wire loop and inoculate into a culture tube containing 50 ml of LB medium supplemented with 500 ml of 20% maltose and 500 ml of 1 M MgSO4 solution. Shake at 160 rpm at 37°C overnight or until the OD600 has reached 0.6. Store the culture at 4°C until use.

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

5. According to the titration data, dilute the IcDNA library with phage buffer. Set up 20 to 25 plates for primary screening of the cDNA library. For each of the 100-mm plates, mix 0.1 ml of the diluted phage containing 2 x 105 pfu of the library with 0.15 ml of fresh bacteria in a microcentrifuge tube. Cap the tube and allow the phage to adsorb the bacteria at 37°C for 30 min.

Note: The number of clones needed to detect a given probability that a low-abundance mRNA is converted into a cDNA in a library is:

where N is the number of clones needed, P is the possibility given (0.99), and 1/n is the fractional portion of the total mRNA, which is represented by a single low-abundant mRNA molecule. For example, if P = 0.99, 1/n = 1/37,000 and the N = 1.7 x 105.

6. Add the incubated phage-bacterial mixture to the tubes from the water bath. Vortex gently and quickly pour onto the center of the LB plate, quickly spreading the mixture over the entire surface of an LB plate by tilting the plate. Cover the plates and allow the top agar to harden for 5 to 10 min in a laminar flow hood. Invert the plates and incubate them in an incubator at 42°C for 4 h.

Note: The top agar mixture should be evenly distributed over the surface of an LB plate. Otherwise, the growth of the bacteriophage may be affected.

7. Saturate nitrocellulose filter disks in 10 mM IPTG in water for 30 min and air-dry the filters at room temperature for 30 min.

8. Carefully overlay each plate with a dried nitrocellulose filter disk from one end and slowly lower it to the other end of the plate. Avoid any air bubbles underneath the filter. Quickly and carefully mark the top side and position of each filter in a triangle fashion by punching the filter through the bottom agar using a 20-gauge needle containing India ink. Cover the plates and incubate at 37°C for 4 to 6 h. If a duplicate is needed, a second filter may be overlaid on the plate and incubated for another 5 h at 37°C. However, the signal will be relatively weaker.

Note: IPTG induces the expression of the cDNA library and the expressed proteins are transferred onto the facing side of the membrane filters.

9. Place the plates at 4°C for 30 min to chill the top agar and to prevent it from sticking to the filters. Transfer the plates to room temperature and carefully remove the filters using forceps. Label and wrap each plate with Parafilm and store at 4°C until use. The filters can be rinsed briefly in TBST buffer to remove any agar.

Tips: Due to the possible diffusion of proteins, it is strongly recommended to process the filters according to the following steps. Storage at 4°C for a couple of days before processing may cause difficulty in the identification of the actual positive plaque in the plates. The filters must not be allowed to dry out during any of the subsequent steps carried out at room temperature. Based on our experience, high background and strange results may appear, even partial drying. If time is limiting, the damp filters may be wrapped in SaranWrap, then in aluminum foil, and stored at 4°C for up to 12 h. It is recommended to process the filters individually during the following steps to obtain an optimal detection signal.

10. Incubate a filter in 10 ml of TBST buffer containing 2 to 3% BSA or 1% gelatin or 5% nonfatty milk or 20% calf serum to block nonspecific protein-binding sites on the filter. Treat the filters for 60 min with slow shaking at 60 rpm.

Note: It is recommended that one dish be used for one filter only and that the facing side of the filter be down. Each filter is incubated with about 10 ml of blocking buffer. Less than 5 ml will cause the filter to dry but more than 15 ml for an 82-mm filter may prevent obtaining optimal results.

11. Carefully transfer the filters to fresh dishes, each containing 10 ml of TBST buffer with primary antibody.

Tip: The antibodies should be diluted at 200 to 10,000X, depending on the concentration of the antibody stock. The antibodies purified with an IgG fraction or with an affinity column usually produce better signals. The diluted primary antibody can be reused several times if stored at 4°C.

12. Wash each filter in 20 ml of TBST buffer containing 0.5% BSA for 10 min with shaking at 60 rpm. Repeat washing twice.

13. Transfer the filter to a fresh dish containing 10 ml of TBST buffer with the second antibody-alkaline phosphatase conjugate at 1:5000 to 10,000 dilution. Incubate for 40 min with shaking at 60 rpm.

14. Wash the filters as described in step 12.

15. Briefly damp-dry the filter on a filter paper and place it in 10 ml of freshly prepared AP color development substrate solution. Allow the color to develop for 0.5 to 5 h or overnight. Positive clones should appear as purple circle spots on the white filter.

16. Stop the color development when the color has developed to a desired intensity in 15 ml of stop solution. The filter can be stored in the solution or stored dry. The color will fade after drying but can be restored in water.

17. In order to locate the positive plaques, carefully match the filter to the original plate by placing the filter, facing side down, underneath the plate with the help of marks made previously. This can be done by placing a glass plate over a lamp and putting the matched filter and plate on the glass plate. Turn on the light so that the positive clones can be easily identified. Transfer individual positive plugs containing phage particles from the plate using a sterile pipette tip with the tip cut off. Expel the plug into a microcentrifuge tube containing 1 ml of elution buffer. Elute phage particles from the plug at room temperature for 4 h with occasional shaking.

18. Transfer the supernatant containing phage particles into a fresh tube and add 20 ml of chloroform. Store at 4°C for up to 5 weeks.

19. Determine the pfu of the eluate, as described in the section entitled Subtracted cDNA Library. Replate the phage and repeat the screening procedure with the antibody probe several times until 100% of the plaques on the plate are positive. (Figure 3.7)

Tips: During the rescreening process, the plaque number used for each plate should be gradually reduced. In my experience, the plaque density for one 100-mm plate in the rescreening procedure is 1000 to 500 to 300, and to 100.

20. Amplify the putative cDNA clones and isolate the recombinant iDNAs by either plate or liquid methods.

Third screening

FIGURE 3.7 Diagram of rescreening and purification of positive clones from a cDNA library.

Third screening

FIGURE 3.7 Diagram of rescreening and purification of positive clones from a cDNA library.

Reagents Needed

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

Adjust pH to 7.5 with 0.2 N NaOH. Autoclave.

LB Top Agar (500 ml)

Add 4 g agar to 500 ml of LB medium and autoclave.

Elution Buffer

10 mM Tris-HCl, pH 7.5 10 mM MgCl2

IPTG Solution

10 mM Isopropyl ß-D-thiogalactopyranoside in dd.H2O

Phage Buffer

TBST Buffer

Blocking Solution

3% BSA, or 1% gelatin, or 5% nonfat milk in TBST buffer

AP Buffer

Color Development Substrate Solution 50 ml AP buffer

0.33 ml NBT (nitroblue tetrazolium) stock solution 0.165 ml BCIP (5-bromo-4-chloro-3-indolyl phosphate) stock solution. Mix well after each addition and protect the solution from strong light. Warm the substrate solution to room temperature to prevent precipitation.

Stop Solution

Method B. Screening a cDNA Library Using 32P-Labeled DNA as a Probe

In addition to the immunoscreening methodology described previously, the cDNA library can also be screened with labeled DNA, which can be labeled by isotopic or nonisotopic methods (see Chapter 7 for detailed protocols), as a probe. The library is screened by in situ plaque hybridization with the probe. Identification of cDNA clones of a potential multigene family can be obtained at relatively low stringency of hybridization. This will be achieved, based on our experience, by reducing the percentage of formamide (30 to 40%) in hybridization solution and washing temperature (room temperature followed by 50 to 55°C) and keeping the temperature of hybridization constant and the salt in the wash solution constant at 2X SSC in 0.1% SDS. To prevent nonspecific cross-linking hybridization, however, a high-stringency condition should be applied. Positive plaques will then be isolated to homogeneity by successive rounds of phage titration and rescreening. The resulting phage lysates will be used in the large-scale preparation of phage DNAs.

The general procedures are the same as those described in the section entitled Screening of a Genomic Library in Chapter 19.

Method C. Screening a cDNA Library Using a Nonradioactive Probe

These general procedures are described in Chapter 19.

Method D. Isolation of lPhage DNAs by the Liquid Method

1. For each 50 ml of bacterial culture, add 10 to 15 ml of 1 x 105 pfu of 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 11 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 bacteriophage used in step 1 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 a long time to see lysis, or no lysis will take place.

3. Immediately centrifuge at 9000 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.

4. Add RNase A and DNase I to the lambda lysate supernatant, each to a final concentration of 2 to 4 mg/ml. Place at 37°C for 30 to 60 min.

Note: RNase A functions to hydrolyze the RNAs. DNase I will hydrolyze chromosomal DNA but not phage DNA that is packed.

5. Precipitate the phage particles with one volume of phage precipitation buffer and incubate for at least 1 h on ice.

6. Centrifuge at 12,000 x g for 15 min at 4°C and allow the pellet to dry at room temperature for 5 to 10 min.

7. Resuspend the phage particles with 1 ml of phage release buffer per 10 ml of initial phage lysate and mix by vortexing.

8. Centrifuge at 5000 x g for 4 min at 4°C to remove debris and carefully transfer the supernatant into a fresh tube.

9. Extract the phage particle proteins using an equal volume of TE-saturated phenol/chloroform. Mix for 1 min and centrifuge at 10,000 x g for 5 min.

10. Transfer the top, aqueous phase to a fresh tube and extract the supernatant one more time as in step 9.

11. Transfer the top, aqueous phase to a fresh tube and extract once with 1 volume of chloroform:isoamyl alcohol (24:1). Vortex and centrifuge as in step 9.

12. Carefully transfer the upper, aqueous phase containing iDNAs to a fresh tube and add one volume of isopropanol or two volumes of chilled 100% ethanol. Mix well and precipitate the DNAs at -80°C for 30 min or -20°C for 2 h.

13. Centrifuge at 12,000 g for 10 min at room temperature and aspirate the supernatant. Briefly wash the pellet with 2 ml of 70% ethanol and dry the pellet under vacuum for 10 min. Resuspend the DNA in 50 to 100 ml of TE buffer. Measure the DNA concentration (similar to RNA measurement described previously) and store at 4 or -20°C until use.

Reagents Needed

SM Buffer (per Liter)

0.01% Gelatin (from 2% stock) Sterilize by autoclaving.

Phage Buffer

20 mM Tris-HCl, pH 7.4 100 mM NaCl 10 mM MgSO4

Release Buffer

Phage Precipitation Solution

30% (w/v) Polyethylene glycol (MW 8000) in 2 M NaCl solution

TE Buffer

TE-Saturated Phenol/Chlorolorm

Thaw crystals of phenol in a 65°C water bath and mix with an equal volume of TE buffer. Allow the phases to separate for about 10 to 20 min. Mix one part of the lower phenol phase with one part of chloroform:isoamyl alcohol (24:1). Allow the phases to separate and store at 4°C in a light-tight bottle.

DNase-Free RNase A

To make DNase-free RNase A, prepare a 10 mg/ml solution of RNase A in 10 mM Tris-HCl, pH 7.5 and 15 mM NaCl. Boil for 15 min and slowly cool to room temperature. Filter if necessary. Alternatively, RNase (DNase-free) from Boehringer Mannheim can be utilized.

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