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RESEARCH DIVISION Laboratory Manual

 


 

cDNA + Genomic DNA Library Screening

  1. Select cells as recommended in the literature accompanying the library. For lgt10 libraries we generally use C600 hfl and for genomic libraries KW251 (Promega, partial genotype = F-, mcrB1, mcrA, hsdR2, recD1014). Prepare an o/n culture in LMM.
  2. Next day pellet the saturated culture and resuspend in half the original volume of autoclaved TM10 (10mM TrisHCl pH7.5, 10mM Mg sulfate).
  3. Titre the library by diluting the phage stock in 100-fold steps in TM10, vortexing gently and briefly to mix (excessive vortexing will damage the phage tails). Cell infection is carried out by adding a dilution of the phage to bacterial cells (150ul of 2x cells for each small plate) in 10ml tubes and incubating at room temperature for 15 minutes. Always include a "no phage" control.
  4. Heat top agarose (0.75% agarose in LMM) to melt and then cool to 45C in water bath. Place the plates in a laminar flow hood to warm slightly and to dry out. The latter is especially important, as free moisture will cause the phage to "run" in droplets and allow the top agarose to pull away if plaque lifts are performed. Properly dry plate should have a slightly crinkled surface.
  5. Add top agarose, flick to mix (avoid bubbles) and add over plate. Allow a minute or so to set and then incubate inverted at 37C.
  6. Calculate the phage titre after 12hr to o/n. Check the size of the plaques after o/n incubation (should be 0.8-1mm) and that no plaques are present on the no phage control plate.

First Round Screen

  1. cDNA and genomic screens are done using 15cm, large plates. The procedure is essentially as above with the following changes:
  2. Use 450l of bacterial cells per sample, 50,000 phage per large plate and twelve plates per screen. Infect the cells as a single pool (ie add phage to 12 X 450l of cells and mix gently), leave at R/T for 15min and then aliquot into 10ml tubes. This ensures each plate is equal density. Add 10-12ml of top agarose, recap and invert twice to mix before pouring. Avoid bubbles as these will displace the filter.
  3. Always use top agarose (not agar) for screens as there is less chance of the plaque layer tearing.
  4. Be very careful to ensure the plates are dry, wet plates are a disaster when it comes to take lifts.
  5. Next day plates should have confluent lysis, best observed against a light. Place at 4C to cool and harden the top agarose. Label nitrocellulose filter with a biro, two sets - A & B - 1-12, for duplicate lifts.
  6. Add first set of filters (A) to plates and key with indian ink (include a • • on one side to help orientate). Ink is often contaminated if it has been used many times. Get a fresh needle and heat the ink in the microwave briefly. If the plates are kept free of contamination they can be used over a period of 1-2mths.
  7. Remove the filters and add to moistened 3MM (but no puddles!) containing 0.5M NaOH, 1.5M NaCl for 5', plaque side up! Transfer to 3MM moistened with 0.5M TrisHCl pH 7.5, 1.5M NaCl for 5' and lastly to 3MM moistened with 2 X SSC, for 5'. Remove to 3MM sheet and airdry in laminar flow hood. Some people perform these steps by flipping filters into a container of each solution but this results in much weaker signals when the filters are probed.
  8. As the first filters are removed, add the "B" set to the plates. Remember to key these at the same position as the "A" set, then repeat the above steps.
  9. Bake the filters at 80 for two hrs. Check the oven after 30' and wipe away condensation form the door. The filters must be baked not stewed!
  10. Label cDNA probe using protocol in this manual.
  11. Prewet filters in 2 X SSC and prehybridize filters in 40ml Aqua hyb in a large petri dish for 2+hr Hybridize overnight in fresh Aqua. hyb. (1.5ml per filter) with 50-100ng of labelled probe.
  12. Wash at desired stringency. Remove filters to sandwich off paper towel (filters are blotted but still moist so can be rewashed) and then place on gladwrap covered X-ray film. Placing the filters with care ie. all - writing "up", in order, with double key marks to the "6-o'clock position - will make it easier to overlay the A and B set of autorads. Include fluorescent markers.
  13. Autoradiograph for 24hr at -70C

Second Round Screen

  1. You should get one to two positives per plate when probing a genomic library for single copy gene, cDNA libraries depend on the abundance of the mRNA you are screening for. Number each of the positives and set up ependorf tubes with 1.0ml of TM10. Pick area from plates corresponding to area of positive phage into 1 ml TM (pick an area of about 2mm dia). Accuracy lining up the key marks is crucial to get all the positives, whilst picking a small area.
  2. Leave at RT 1hr+. Can gently vortex to increase phage dispersion.
  3. Infect 150 ul cells with 1 ul of 1 in 200 dilution of phage in TM, plate on small plate. Take lifts as above (duplicates are not required) and hybridize with same probe.

Third round

  1. Pick positives from second round into 1.0ml TM10 and plate at 1ul of 1:5 for several plaques picked, 1ul for one plaque picked. This screen should achieve single positives plaques separated by >2mm from adjacent plaques so a pure stock can be prepared.

NOTE.     Second and third round screen steps can be shortened: Filters baked for 10' in autoclave, 5' prehyb and hyb during day (6hr).

Lifts From Bacterial Colonies

  1. Label nitrocellulose and carefully place into agar. Mark orientation by piercing through nitrocellulose and agar with needle and ink.

        Set up 3 trays with 3MM paper.

  1. 0.5M NaCl / 0.5M NaOH (Denaturing solution- see Reagents)
  2. 0.5M Tris HCl / 1.5M NaCl (Neutralising solution- see Reagents)
  3. 2 x SSC
  1. Moisten the 3MM paper with appropriate solution. Tip off so that there are no puddles.
  2. Gently lift nitrocellulose from the plate. Apply nitrocellulose (bacteria side up) to the 3MM paper. Expose to each tray for 5 minutes
  3. Blot with paper towel. Sandwich between 3MM paper
  4. Bake for 2 hours at 80C
  5. Return plates to 37C incubator for 2 hours and then store at 4C. 
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998