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DNA ISOLATION AND PLASMID PURIFICATION

> Phenol extraction and ethanol precipitation.

   

> Large Scale Plasmid Prep for 100 ML Superbroth Cultures

   

> Plasmid Miniscreen Protocol (Boil Prep)

   

> Alkaline Lysis Miniprep

   

> Alkaline Lysis Miniprep Modified 1

   

> Alkaline Lysis Miniprep Modified 2

   

> Amplification of plasmids with chloramphenicol

   

> Single-Strand DNA Prep. Large Scale

    > Titre of Bacteriophage
  > Large Scale Bacteriophage Prep

Titre of Bacteriophage

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  1. Plate 50l of SURE cells directly onto an LB plate containing tetracycline. Incubate at 37C overnight.
  2. Pick a colony and onoculate into 5mL LB plus TET. Incubate To saturation at 37C.
  3. Make serial dilutions of stock bacteriophage.
  4. Add 10uL of the appropriate phage dilutions to labelled 15mL Falcon tubes.
  5. Add 0.3mL if saturated E. Coli to each of the phage suspensions. Let sit at room temperature for 20 min.
  6. Move tubes to a 37C waterbath or heat block for 10 min. At this time also melt some TOP agar in the microwave under the defrost setting for several min. or until completely melted.
  7. Let TOP agar cool at room temp. For about 5 min.
  8. Keep melted agar in a water bath at 45-50C.
  9. Take 3mL of the TOP agar and add to the E. coli/phage mixture. Quickly vortex and pour the contents of the tube onto a pre-warmed LB plate. Tilt the plate so as to get even distribution of the melted agar. (This step must be done quickly so that the agar doesn't solidify prematurely).
  10. Do this for each of the phage dilutions and then place the plates into the 37C incubator for 10-12 hours.
  11. A plague can be picked from the plates which contain less than 100 plaques. Only plaques should be picked which have neighbouring plaques more than 1 cm away.