Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
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cDNA Library Titering, Screening, and Clone Isolation |
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| Contributor: |
The Laboratory of Donald Rio at the University of California, Berkeley | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Overview |
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| This protocol describes colony hybridization and screening of a cDNA library propagated as plasmids in bacteria. This procedure will prove useful when the complexity of the genes represented in the library is small (e.g., Saccharomyces cerevisiae genes), or when the abundance of the gene of interest is great (e.g., Actin). Phage libraries are recommended for more complex or lower abundance gene screening. This protocol also describes an innovative step for accomplishing the lysis of bacteria on nylon membranes with an autoclave. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Procedure |
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A. Transforming and Plating of the Library 1. Dilute 200 ng of the library to 50 μl with TE Buffer (see Hint #2). Dispense 10 μl into five round-bottom 13 ml polypropylene tubes (Falcon) and allow them to chill on ice. For a negative control, use 40 pg pUC19 plasmid DNA or equivalent vector. 2. Thaw three tubes (600 μl) of DH5-α competent cells on ice. 3. Aliquot 100 μl of competent cells into each tube containing DNA. 4. Incubate the tubes on ice for 30 min. 5. Heat-shock the cells at 42°C for 45 sec. Do not allow the tubes to stay at 42°C for longer than 45 sec. 6. Return the tubes to ice and incubate for 2 min. 7. Add 900 μl of SOC to each tube and incubate at 37°C for 1 hr with shaking. 8. Combine all of the transformed cells into one 15 ml conical tube. Add 800 μl of sterile 10% Glycerol. Dispense 500 μl aliquots of the transformed cells into cryovials. 9. Place 10 μl of transformed cells into a separate vial for titering. For the control plasmid transformation, add sterile Glycerol to 1.5 % (v/v). 10. Freeze all the tubes in a Dry Ice/Ethanol bath. Make sure that the Ethanol does not rise above the cap line. Store the vials at -80°C. 11. Thaw the 10 μl aliquot and the control transformants on ice. Make dilutions into SOC. For the control plasmid, make 1:100 and 1:1,000 dilutions. For the library, make 1:100 through 1:100,000 dilutions in 10-fold increments (4 dilutions total). 12. Plate 100 μl of each dilution onto LB plates with Amp. 13. Place plates at 37°C overnight. 14. To calculate the colony forming units (cfu) value per ml of culture, multiply the number of colonies by the dilution factor/the volume of culture (ml) plated. 15. The following steps require twelve to fourteen 25 cm LB plates with Amp and six to seven LB/Glycerol plates: Approximately 40,000 to 50,000 clones per plate are required (see Hint #3) Spread on 6 or 7 plates When a good titer is determined, dilute enough of the transformation into SOC such that about 500 μl contains 40,000 to 50,000 clones 16. Place a nylon filter onto an LB plate with Amp and wet completely. Add 500 μl of diluted transformed cells and spread evenly. Repeat this for all the plates. 17. Set the plates at 37°C until the colonies start to appear. This will require approximately 7 to 8 hr. They should be no larger than a period at the end of a printed sentence. 18. Remove the plates from the incubator and prepare replica filters as follows: a. Place a glass plate on the bench. Place a 1 MM Whatman filter paper on the plate. Place the master filter (lifted directly for the LB plate), colony side up, onto the filter paper. Very carefully, place a second filter on top of the master filter. Do not move the second filter once it has been placed. Place a piece of 1MM Whatman paper on the top, followed by another glass plate. b. Press evenly on the top of the sandwich to transfer some of the colonies onto the replica filter. c. After pressing, carefully remove the top glass plate and top piece of filter paper. d. Carefully punch holes with a sterile 18-gauge needle from the replica filter through the master filter in an asymmetric, easy to identify pattern (i.e., 1 hole, 2 holes, and 3 holes spaced at 120 degree intervals just inside the circumference). e. Remove the replica filter and place it colony side up onto a fresh LB plate with Amp. f. Add a second filter on top of the master filter and finish the sandwich as before. Press to transfer the colonies, then remove the glass plate and top Whatman paper as before. g. To make the same pattern of holes as before, turn the filters over so that the master filter is on top. The holes should be clearly visible. Poke through the old hole on the master filter with the same needle through the second replica. h. Once finished, peel apart the filters and place the replica filter onto another LB plate with Amp and replace the master filter on a LB/25% Glycerol plate with Amp. i. Repeat this process for all of the filters, changing the Whatman paper for each filter. 19. Wrap Parafilm around the master plates and store them at -20°C. 20. Working quickly from this point, place all replica filters onto 3 MM Whatman filter paper. Be careful not to let the filters dry. Place into autoclave trays. 21. Autoclave for 1 min at 220°C with a fast-exhaust program to lyse the cells. 22. Let the filters air-dry overnight at room temperature. B. Screening the library 1. To denature the DNA from the lysed colonies, soak the filters in 500 ml of 0.4 M NaOH at 42°C for 30 min. 2. Soak the filters in 500 ml of Wash Buffer 1 at 42°C for 30 min. 3. Prehybridize the filters in 25 to 30 ml of Prehybridization Solution at 42°C for 4 to 8 hr. 4. Add 25 ml of fresh Prehybridization Buffer and denatured probe (see Protocol ID#916 or Protocol ID#566). 5. Hybridize overnight at 42°C. 6. Rinse away excess probe by immersing the filters in a solution of 2X SSC/0.1%SDS for a few seconds and begin washing the filters with gentle agitation immersed in the following solutions sequentially: a. 2X SSC/0.1% SDS twice at 68°C b. 0.5X SSC/0.1% SDS twice at room temperature c. 0.1X SSC/0.1% SDS twice at room temperature Check the filters with a Geiger counter after each drop in salt concentration wash (see Hint #4). 7. Put fluorescent pen marks or radioactive-inked marks in the cassette so you can align the film with the filters. 8. Expose the filters to film at -80°C for 2 to 3 days. 9. Circle positive colonies on the film with a Sharpie marker. Additionally, mark the pinhole sites on the film. 10. Set up enough tubes with SOC in advance for picking all the positive clones. Have a flame and loop ready for picking the clones. 11. Working quickly, remove one master plate from the freezer. Remove the lid and lift the filter off of the plate with blunt filter forceps. Place the filter, colony side up, onto the Petri dish lid. Line up the pinholes on the film with those on the filter. Perform this on a light box. 12. Once aligned, scrape the positive clone off the filter with the (flamed and cooled) inoculation loop. Obtain a fairly large area to ensure that the clone was gathered. Wash the cells into a tube containing 3 ml SOC and flame the loop. Repeat this process for all of the positives on the filter. 13. Once finished with a filter, place it onto a freshly made LB/Glycerol plate with Amp. Wrap the plate in parafilm and place it back at -20°C. Proceed with the next filter. 14. Grow the clones at 37°C for 2 to 3 hr. 15. To rescreen the clones, plate about 1,000 colonies on a 100 mm plate. From a more dense culture, plate 100 μl of 1:100 and 1:1,000 dilutions onto LB plates with Amp. From a less turbid culture, plate 100 μl of undiluted and 1:100 diluted culture. Place the plates at 37°C overnight. 16. Add Glycerol to the liquid cultures to 15% (v/v) and store at -80°C. 17. Perform colony lifts from the plates with the most optimal density of colonies and repeat hybridization. 18. Perform additional rounds of rescreening, with about 100 colonies per plate, until a single, well-isolated colony can be picked for each positive clone. |
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| Solutions |
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| BioReagents and Chemicals |
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Bovine Serum Albumin (BSA), Fraction V Glycerol Ficoll 400 Dry Ice Dextran Sulfate Sodium Hydroxide EDTA Sodium Citrate Yeast Extract Tryptone Magnesium Chloride Tris Ethanol Potassium Chloride Sodium Chloride Formamide Polyvinyl Pyrolidone Sodium Phosphate SDS Denatured DNA DH-5-alpha competent cells (BRL) Magnesium Sulfate Glucose Ampicillin |
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| Protocol Hints |
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1. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions. 2. Library DNA should be stored at -80°C in TE buffer at 0.1 μg/ml. 3. Since cells will be plated directly onto nylon filters, the number of cells that will need to be plated will be much greater than if plated without the filters. It is advisable to grow a small plate with a filter present in order to account for the decrease in survivability when cells are plated on filters. 4. After a 0.5X SSC/ 0.1% SDS wash at room temperature, the background will usually be acceptable; but this should be determined empirically. Stop once the background sounds good, or the filters will need to be exposed for an extended period of time. |
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