DNA

Part:BBa_K4636053:Design

Designed by: Lo-Chueh, Chu   Group: iGEM23_NTHU-Taiwan   (2023-10-11)

insert_ligation_0004771


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design

For two restriction sites design, we should consider following tips[1], [2], [3] :
(1) Sitting sequence (4~5 b.p.) : Located outside of the restriction site and responsible for enzyme activity. We should avoid the same sequence as restriction sites, and it must not contain GG or CC sequence.
(2) Restriction sites can’t be found in insert.
(3) Use two different restriction sites with distinct restriction enzymes.
(4) The two different restriction enzymes should have similar reaction temperatures and use the same reaction buffer.
(5) Both of the restriction enzyme have high activity in the same reaction buffer.

Reference

1. https://international.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
2. https://www.addgene.org/protocols/primer-design/
3. https://www.researchgate.net/post/Are_the_sitting_sequences_added_before_the_PCR_primers_arbitrary_or_specific_to_restriction_endonucleases