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TargeTron
gBlock

LtrB TargeTron

Retargeted fragment of a TargeTron is constructed by PCR or ordered as a gBlock Gene Fragments from IDT, digested with appropriate enzymes (e.g. HindIII and BsrGI) and swapped for the corresponding fragment of a donor plasmid.

Set up the PCR reaction as follows:

       4.4 μl of water
       4.0 μl of PCR buffer (5X)
       0.5 μl of dNTPs (10 mM)
       2.0 μl of MgCl2 (25 mM)
       1.0 μl of the LtrB targetron template (20 ng)
       2.0 μl of the LtrB primer mix
       2.8 μl of IBS1/2 primer (10 μM)
       2.8 μl of EBS1/δ primer (10 μM)
       0.5 μl of DNA polymerase (1-2.5 U)

Cycle the PCR reaction as follows:
  1. 94oC, 1 minute (Initial Denaturation)
  2. 94oC, 30 seconds (Denaturation)
  3. 55oC, 30 seconds (Annealing)
  4. 72oC, 24 seconds (Extension)
  5. Go To Step 2, 29 times
  6. 72oC, 2 minutes (Final Extension)
  7. 4oC (Soak)

EcI5 TargeTron

Retargeted fragment of a TargeTron is constructed by PCR or ordered as a gBlock Gene Fragments from IDT, digested with appropriate enzymes (e.g. XbaI and AvaII) and swapped for the corresponding fragment of a donor plasmid.

Set up the PCR reaction as follows:

       4.4 μl of water
       4.0 μl of PCR buffer (5X)
       0.5 μl of dNTPs (10 mM)
       2.0 μl of MgCl2 (25 mM)
       1.0 μl of the EcI5 targetron template (20 ng)
       2.0 μl of the EcI5 primer mix
       2.8 μl of IBS1/2S primer (10 μM)
       2.8 μl of EBSR primer (10 μM)
       0.5 μl of DNA polymerase (1-2.5 U)

Cycle the PCR reaction as follows:
  1. 94oC, 1 minute (Initial Denaturation)
  2. 94oC, 30 seconds (Denaturation)
  3. 55oC, 30 seconds (Annealing)
  4. 72oC, 36 seconds (Extension)
  5. Go To Step 2, 29 times
  6. 72oC, 2 minutes (Final Extension)
  7. 4oC (Soak)