Difference: ProtocolsSsdnaRecombination (5 vs. 6)

Revision 62013-03-28 - DanielDeatherage

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META TOPICPARENT name="ProtocolList"

λ red mediated ssDNA gene modification

Background

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      • Mutation Oligo 1: bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb RR M RR bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb
    • Mutations MUST be placed on the lagging strand.
      • For E. coli REL606 (and other B strains) origin of replication is at ~10:00 not 12:00 (3886105-3886336). Therefor:
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        • If mutation location between ~ 3,886,500 and ~ 1,571,300 : design against reverse strand
        • If mutation location between ~ 1,572,000 and ~ 3,886,000 : design against forward strand
        • If mutation location between ~ 3,886,000 and 3,886,500 or ~1,571,300 and 1,572,000 it's probably best to try both strands and see which is better. Expect 100+ fold increase in efficancy for lagging strand.
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        • If mutation location between ~ 3,886,500 and ~ 1,279,677 : design against reverse strand
        • If mutation location between ~ 1,661,443 and ~ 3,886,000 : design against forward strand
        • If mutation location between ~ 3,886,000 and 3,886,500 or ~1,279,500 and 1,662,000 it's probably best to try both strands and see which is better. Expect 100+ fold increase in efficancy for lagging strand.
 
  1. Mutation Screen Oligo:
    • Primers used for screening colonies for presence of transformants
    • Should be ~20bp long, amplicon of ~150+ bp, and one primer must have 3' bases as mutations of interest.
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  -- Main.DanielDeatherage - 25 Mar 2013
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