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DNA
Part:BBa_K4636055:Design
Designed by: Lo-Chueh, Chu Group: iGEM23_NTHU-Taiwan (2023-10-11)
pUC19_insert_ligation_0004771
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
Consideration of primer for amplifying insert_ligation by PCR.[2]
(1) Length : 18~24 base pairs.
(2) GC content : 40%~60%.
(3) 3’ end with highly GC.
(4) Tm : 50~60°C.
(5) Choosing a sequence that makes the primer highly specific and can not anneal to other sites.
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