Restriction Enzyme Cloning

Restriction enzyme cloning is a bread-and-butter technique in molecular biology for modifying plasmids to contain genes or other DNA sequences of interest. While it may be more time consuming than some recently developed techniques, it is very reliable.

For background on restriction enzyme cloning and some pretty pictures, check out the wiki on the topic, and check out the useful diagram on this Chinese website.

Materials

Plasmid Marker
pJEB11 →Ara
pJEB12 →Ara+
pJEB15 →Mal

  • Cloning plasmid (such as pUC19) and DNA fragment to be cloned
  • Restriction Enzymes
  • DNA Ligase
  • Electrocompetent cells or chemically competent cells of an appropriate cloning strain.
  • Antibiotic Plates to your cloning plasmid and/or the antibiotic resistance gene you are cloning
  • XGal and IPTG (for blue/white screens)

Step 1: Design Primers

  1. Thaw one 1.7 ml microfuge tube of electrocompetent cells for each parent strain on ice.
  2. While waiting for this to thaw, place one electroporation cuvette on ice for each parent strain.
  3. Add 1 l of pACBSR and 1 l of the donor plasmid to each tube of electrocompetent cells on ice. Mix by flicking the tube with your finger. Do not pipette up and down or vortex to mix.
  4. Electroporate
  5. Pipette sample out of cuvette into original microfuge tube on ice. Add 500 l of room temperature SOC medium.
  6. Grow at 37C for 1 hr in a shaking incubator to induce antiobiotic expression.
  7. Plate 100 l and 10 l + 90 l saline on two separate LB + Cam + Kan plates.
  8. Grow plates overnight at 37C.

Step 2: Perform PCR on Template to amplify desired product with restriction sites

  1. Pick three colonies from each successful transformation into separate test tubes containing 5 ml of LB medium supplemented with 0.2% L-Arabinose and 20 g/ml Chloramphenicol (Cam). The arabinose is to induce expression of the λ Red genes from the gene-gorging plasmid. Use arabinose regardless of which sugar marker you are changing.
  2. Grow cultures overnight at 37C, shaking at 120 rpm.

Day 3: Screen or Select for Desired Mutation

  1. Make a 104-fold dilution of each overnight culture with two 100 l transfers through dilution tubes containing 10 ml of saline. Note that the cell density achieved after induction is considerably lower than that usually achieved during growth in LB.
  2. A. If you are gene gorging the version of the the marker you must screen for successful mutations. Plate 200 l of a 104 dilution of each culture on tetrazolium arabinose (TA), tetrazolium maltose (TM), or other suitable indicator agar containing 20 g/ml Chloramphenicol (Cam). Successful mutants will be red.
    B. If you are gene gorging the + version of the the marker you could screen for successful mutations as above, but it is easier to select for them by plating 100 l of a 102 dilution on minimal arabinose (MA) or minimal maltose (MM).
  3. Grow plates overnight at 37C.

Day 4: Screen for Gene-Gorging Plasmid Loss

Expected results are 0-10 colonies with the marker change per 200 colonies of the other color.

  1. Pick one colony from each plate into 5 ml of LB medium in a test tube. Give each picked colony an isolate designation. Colonies from the same plate are not necessarily independent isolates, they may share undesired second-dite mutations. Picking individual isolated
  2. Grow cultures overnight at 37C, shaking at 120 rpm.
  3. Grow plates overnight at 37C.

Day 5: Plate to Single Colonies

  1. Plate 100 l of a 106 dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe). The dilution can be made with three 100 l transfers through dilution tubes containing 10 ml of saline.
  2. Grow plates overnight at 37C.

Day 6: Patch for Plasmid Loss

  1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with 20 g/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic.
  2. Grow plates overnight at 37C.

Day 7: Save a Freezer Stock of the New Strain

  1. Pick from a patch that grows without antibiotic and not with either antibiotic present. Usually a majority of patched colonies have successfully lost both plasmids.
  2. Grow cultures overnight at 37C and archive 2 × 1 ml frozen copies in 10% glycerol.

Test for Marker Neutrality

Competitive fitness assays should be used to test for non-neutral mutations that sometimes occur during strain construction by this method. Testing three independent isolates usually assures one success.

References

  1. Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311, 153-163.
  2. Sean Sleight's Detailed Procedure.

-- Main.MichaelHammerling - 01 Feb 2012

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Topic revision: r1 - 01 Feb 2012 - 19:35:58 - Main.MichaelHammerling
 
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