Misc Notes

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Domestication:

Once you determine which Type IIS RE you will use for your cloning, be sure to check your inserts in silico for the recognition sites (RS) for all the Type IIS RE you will use. If you have a recognition site in your insert, use this flow chart to determine your best course of action:
  1. What are the overhangs that the cut would leave?
    • An overhang that is present in my system
      • If the overhang left behind is one that could ligate with one of the overhangs left behind by your system, it would be best to domesticate it 
        • proceed to #3
    • An overhang that is not present in my system
      • If the overhang left behind is not in your platform's syntax (meaning it would be the only overhang of its type created by the reaction), it may not be of much concern. This will decrease your cloning efficiency, but as long as you are aware of it and you screen for it, you can often getaway without domesticating it
        • go ahead and try your reaction first
  2. Do you have multiple RS do you have in your insert?
    • If you have 2+ RS in your insert, I would proceed to domestication



  3. How to domesticate 
    • Domestication is the elimination of a a RS from your insert. This is typically done by altering a nucleotide to maintain the amino acid sequence

    • 1 RS 
      • Simply use a primer to alter one of the nucleotides so that the amino acid sequence is maintained and the RS is eliminated

    • 2-4 RS 
      • See if you can use a primer or two to fix the problem, but if you have to do more than 3 PCRs, I would consider synthesizing your fragment with the desired changes

    • 4+ RS
      • In my opinion, I would synthesize anything with 4+ RS to save on manhours 


    • Domestication in CDS

      • If the gene is both from a plant genome and is intended for a plant genome, you may be able to get away without having to isolate/validate the domestication in the CDS before your final transformation 

      • If the gene is not from a plant, you should validate the domestication in planta 

    • Domestication in promoter, signal peptide, or terminator 

      • Any domestication in an element outside of CDS is recommended to be validated in your system








Modular Cloning Jargon and Concepts
Restriction Enzymes for Modular Cloning
GoldenGate Overview
GoldenGate Options for Plant Science
Setup and Staying Organized
Misc. Notes






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