Part:BBa_K4636052

insert_ligation_0101802

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]

Main

Insert_ligation_0101802 is a product from ligation between Insert_0101802 and Insert_0101802 itself.(BBa_K46360040)(See Figure 1 for details)

As mentioned on our design page, in order to make long tandem DNA that are continuously repeated cDNA of 0101802, we perform insert ligation, ligate the insert with the insert itself, then transfer it to pUC19 vector and transform into the bacteria. As long as we use primer outside of insert_ligation, we can carry out PCR amplification and obtain a large amount of linear, continuously repeated long tandem of DNA. After that, we could make single-stranded long-term repeated DNA by asymmetric PCR.

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.

Experiment

In Figure 2, the presence of multiple bands with equal intervals in Lane 3 suggests the successful ligation of single-piece inserts into a long-repeated insert sequence with varying lengths.

Figure 2. The agarose gel electrophoresis result of insert ligation. Lane 1: 1 kb DNA ladder, Lane 2: Insert_0004771 ligation product, Lane 3: Insert_0101802 ligation product.

The digested long-repeated insert (Insert_0101802, BBa_K46360040) sequence have successfully ligated into the pUC19 vector. In Figure 3, a band with approximately 4366 bp (vector 2686 bp + long-repeated insert sequence 1680 bp) indicates the successfully ligation process.

Figure 3. The agarose gel electrophoresis result of plasmid. lane 1: 1kb DNA ladder. lane 2: pUC19 vector only. lane 3: pUC19 vector ligated with long-repeated insert sequence

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