Part:BBa_K4636004

PCR_0101802rv

PCR_0101802rv (BBa_K4636004) is a reverse primer for Polymerase chain reaction (PCR) reaction. This primer is designed to amplify Insert_0101802. Sequence and Features

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]

This primer includes a restriction site, sitting sequence, annealing site, and random sequence, all of which can be utilized in subsequent reactions in our project (Figure 1).

Figure 1. Our design for PCR_0101802fw.

We use this primer to amplify our Insert_0101802 (BBa_K46360040) with polymerase chain reaction (PCR).

Design

We obey the standard PCR primer design consideration and used Primer 3[1] to search the suitable site then check it by IDT OligoAnalyzer™ Tool. [2]

Standard PCR primer design consideration :.

(1) Length : 18~24 base pairs.
(2) GC content : 40%~60%.
(3) 3’ end with highly GC content.
(4) Tm : 50~60°C

For restriction site sequence design, we refer to previous study [3], [4], [5]:

(1) Restriction site : To ligate with pUC19 vector, we design restriction site BamH1.
(2) Sitting sequence (4~5 b.p.) : Before restriction site and it’s for enzyme activity. Rule : Avoid the same sequence as RE and have no GG or CC sequence.
(3) Annealing site : Complementary to Insert_0101802 (BBa_K46360040).
(4) Random sequence : Adjust Tm value of primer.

Experiment

We used the PCR_0101802fw and PCR_0101802rv primers to amplify Insert_0101802. As demonstrated in the gel electrophoresis results (Figure 2), the band's correct position confirms the accuracy of our primer design.

Figure 2. PCR result of Insert_0101802. Lane 1: 50bps ladder, Lane 2,3: Insert_0101802
Since the digestion site is included in our PCR primers, a successful digestion of the insert(Figure 3. lane 3) amplified by PCR_0101802fw and PCR_0101802rv would indicate the success of the primer design.

Figure 3. The agarose gel electrophoresis result of insert digestion. lane 1: 100 bp DNA ladder. lane 2: Insert_0004771 digestion product. lane 3: Insert_0101802 digestion product.

Reference

1. https://primer3.ut.ee/
2. https://sg.idtdna.com/pages/tools/oligoanalyzer
3. https://www.addgene.org/protocols/primer-design/
4. https://international.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
5. https://www.researchgate.net/post/Are_the_sitting_sequences_added_before_the_PCR_primers_arbitrary_or_specific_to_restriction_endonucleases