Difference: ProtocolsPsltsEditing (6 vs. 7)

Revision 72015-05-01 - DanielDeatherage

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META TOPICPARENT name="ProtocolList"

Overview

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  1. Grow overnight at 30C.
  2. Resuspend an entire colony in 500l saline.
    • TIP Original protocol calls for PBS not saline. Saline used instead as thought to be unlikely to effect anything, and makes use of existing stock solutions.
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    • Frequency of Double stranded break repair reported at 1 in 105
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  1. Plate 50-100l of resuspended colony onto LB plates containing Carbenicillin and anhydrotetracycline (100ng/mL).
    • Frequency of Double stranded break repair reported at 1 in 105. If no colonies are obtained, consider replating at higher density and/or checking the density of cells actually plated by plating on LB Carbenicillin plates without anhydrotetracycline.
    • If frequency appears lower than expected, efficency can be increased by a pre induction of the λ-Red recombinase. These are optional steps that should not be necessary.
      1. Resuspend a single colony containing the integrated mutation cassette in 50l of DNase-free water.
      2. Confirm correct integration of mutation cassette by PCR.
        1. 20l used as template
        2. 100C incubation prior to amplification.
        3. Amplification should be done using forward primer of 5' mutation cassette and reverse primer of the 3' mutation cassette.
      3. The remaining 30l was mixed with 120l of saline. TIP again, note PBS suggested in original protocol.
      4. 50l plated on LB Carbenicillin plates with or without anhydrotetracycline (100ng/mL).
      5. Remaining 50l used to inoculate 1mL LB + Carbenicillin media.
      6. Incubate 1hr 30C with shaking.
      7. Add L-Arabinose to final concentration of 2mM. TIP see note above about variability in amount of L-Arabinose used in induction.
      8. Incubate 2hr 30C with shaking.
      9. 50l plated on LB Carbenicillin plates with or without anhydrotetracycline (100ng/mL).
    • 5-10 colonies from each plate patched onto LB and LB + Kanamyocin plates.
 

iDT "Gene-block" based

Reference

 
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