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Method: Transformation of Plasmids/Cosmids into E. coli

April 13 1990

Matthew S. Holt


Purpose:

Time required:

  1. Transformation - 1 hour
  2. Growth - approximately 16 hours for visible colonies

Special Reagents:

  1. Competent Cells
  2. SOB Media (needed for DH5-ALPHA cells only)
  3. SOC Media (needed for DH5-ALPHA cells only)
  4. X-gal (needed for color selection only)
  5. dimethylformamide (needed for color selection only)
  6. IPTG (needed for color selection only)

Preface:

Procedures:

Transformation of intact plasmid/cosmid:

Day 1

  1. Verify selection sequence for your plasmid/cosmid (usually ampicillin resistance but not always). You will need LBM plates with this antibiotic.

  2. Place 5-10 ng of plasmid/cosmid DNA into a labeled sterile eppendorf tube. Have an empty tube labeled as a negative control. Competent cells cannot grow on antibiotic plates without a plasmid/cosmid carrying the resistance gene so the negative control plate should not grow colonies. If possible as a positive control use 1-5 ng of a plasmid (e. g. pBR322) that previously has transformed this batch of competent cells efficiently.
  3. Place competent cells directly on ice after removing from -80 degrees C storage. As the cells thaw add 100 ul to each eppendorf tube. Flick the tubes to mix contents and place immediately on ice for 30 minutes.

  4. Remove tubes from ice and incubate for 2 minutes in a 37 degrees C waterbath for a heat shock. The heat shock makes the cell close its "pores" and retain the plasmid/cosmid DNA. Add 900 ul of sterile 1X LBM (or SOC for DH5- ALPHA) to each tube and continue incubating at 37 degrees C for 30 minutes.

  5. Because intact plasmids/cosmids transform efficiently (approximately 1x10e7 per ug of supercoiled DNA) you may wish to plate two dilutions of each to ensure isolated colonies. Take 10 ul and 100 ul of each culture and plate on LBM+ Amp plates (if ampicillin is the selection) using a glass spreader. The 100 ul plates should be quite dense. Flame the spreader between plates and allow to cool before using. Give the plates 5 minutes to absorb the inoculum then invert and incubate at 37 degrees C for 16 hours or until colonies are of the desired size. Store the remainder of the transformation mixture at 4 degrees C. (If necessary it can be reused for up to one week.)

Day 2

  1. Examine the plates and determine the efficiency of transformation.

  2. Pick isolated colonies to prepare miniprep DNA. Quantitate and verify plasmid/cosmid DNA with the appropriate restriction enzyme digests and 1kb ladder and lambda standards. If large amounts are needed follow the procedure for a large scale plasmid preparations when the plasmid/insert is verified.

Transformation of ligation mixes:

Day 1

  1. Verify selection for your plasmid/cosmid. You will need LBM plates with this antibiotic.
  2. Pipet 1/2 of the prepared ligation mixture and two controls (see ligation protocols) into eppendorf tubes. You may wish to plate competent cells alone on a selection plate as a negative control. The remainder of the ligation mix should be stored at -20 degrees C and used as a back-up if necessary.
  3. Place the competent cells directly on ice after removing from the -80 degrees C freezer. As the cells thaw add 100 ul to each eppendorf tube. Flick the tubes to mix and place immediately on ice for 30 minutes.
  4. Remove tubes from ice and incubate for two minutes at 37 degrees C in a waterbath for a heat shock. Add 900 ul of sterile 1X LBM (or SOC for DH5-Alpha) to each tube and continue incubating at 37 degrees C for 30 minutes.
  5. The efficiency of tranformation depends on the ligation mix so you may wish to plate 100-200 ul or several plates of 200 ul for each ligation. Plate 100-200 ul of each on LBM+ antibiotic (SOB+ antibiotic for DH5- Alpha) using a glass spreader. This amount is usually sufficient to obtain the isolated colony. Flame the spreader between plates and cool before each use. After inoculum has been absorbed (5 minutes) invert plates and incubate at 37 degrees C for 16 hours or until colonies are of the desired size. Store remaining transformation culture at 4 degrees C. This can be reused if necessary for up to one week.

Day 2

  1. Examine the plates and determine the efficiency of transformation.
  2. Pick isolated colonies to prepare miniprep DNA. Quantitate and verify plasmid/cosmid DNA with the appropriate restriction enzyme digests and 1kb ladder and lambda standards. If large amounts are needed follow the procedure for a large scale plasmid preparations when the plasmid/insert is verified.

Color selection:

Solutions:

  1. SOB Media:

    2% Bactotryptone 0.5% Yeast extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2 10 mM MgSO4 1.5% Agar (for plates)

  2. SOC Media:

    SOB + 20 mM Glucose
  3. X-gal:

    Dissolve 100 mg of X-gal in 5 ml of dimethylformamide in a sterile polypropylene tube. Aliquot 1 ml into eppendorf tubes wrapped in foil  (to prevent damage by light) and store at -20 degrees C.  It is not  necessary to filter sterilize X-gal solutions.

  4. IPTG:

    Dissolve 2 g of IPTG in 8 ml  of dH2O in a sterile polypropylene tube.  Adjust the volume to 10 ml  with dH2O and filter through a 0.22 micron syringe filter into 1 ml aliquots and store at -20 degrees C.

References:

Sambrook J. Fritsch E.F. and T. Maniatis (1989). Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press p.1.74.