Part:BBa_K4636055

pUC19_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]

Main

As mentioned on our design page, in order to make long tandem DNA that are continuously repeated cDNA of 0004771, we perform insert ligation, ligate the insert with the insert itself (BBa_K46360041), 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.

In our project, we digest this pUC19 vector with BamHI and HindIII site, and then ligate insert_ligation_0101802 (BBa_K46360053) . Then, we transform it into DH5α-E.coli, select colony, and run the colony PCR to check whether we successfully do the cloning.

Figure 1. puc19_insert_ligation_0004771 contain 5 times repeated insert_0004771 and pUC19 vector.

pUC19 vector (Figure 1) is a commonly used vector which conveys ampicillin resistant gene for further selection. Here are some features according to NEB website[1].
(1) It’s length (2686bps) is smaller than most of the plasmid.
(2) pUC19 vector has high copy number which makes it a good candidate to be used in our project.
(3) pUC19 vector contains a 54 bps multiple cloning site polylinker that has 13 restriction sites.

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.

Experiment

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

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.

Due to time and resource constraints, we haven't ligate the long-repeated insert (Insert_0004771, BBa_K46360041) into pUC19 vector.

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