Primer
SDM2_Fw

Part:BBa_K4417003

Designed by: Jiaying Zou   Group: iGEM22_UCL   (2022-09-29)


SDM2 Forward Primer for pCT5c Mutation

Description

This primer is designed for mutating the BsaI site 2 in pCT5-bac 2.0. This part is a forward primer with the same sequence as the original plasmid.

Figure 1: pCT5-bac 2.0 with primers for site directed mutagenesis at position 6004.


Usage and Biology

  • This part is used in site directed mutagenesis of pCT5c (Part: BBa_K4417000).
  • Sequence: ggctccagatttatcagcaataaaccagcc
  • Length: 30-mer
  • Tm: 63℃
  • GC content: 47%


Figure 2: Binding site of SDM2_Fw.

Method

This part is used with BBa_K4417000 and BBa_K4417004. A detailed protocol is described in BBa_K4417000.

Characterization

After SDM1, this primer will remove one BsaI site, leaving only one BsaI site. The KLD product was transformed into DH5-α, and the plasmid was checked using a diagnostic digest. From Figure 3, the expected band size can be identified in lanes 4, 8, 12, and 16.

Figure 3: Comparative gel for pCT5c site directed mutagenesis; 1: HyperLadderTM 1kb, 2: pCT5-bac 2.0 uncut, 3: SDM1 uncut, 4: SDM1,3 uncut, 5: pCT5c uncut, 6: pCT5c cut with BsaI (3481bp, 2344bp, 2052bp), 7: SDM1 cut with BsaI (4396bp, 3481bp), 8: SDM1,3 cut with BsaI (7877bp), 9: pCT5c cut with BsaI, 10: pCT5-bac 2.0 cut with BamHI/SacI (6851bp, 1026bp), 11: SDM1 cut with BamHI/SacI (6851bp, 1026bp), 12: SDM1,3 cut with BamHI/SacI (6851bp, 1026bp), 13: pCT5c cut with BamHI/SacI (6851bp, 1026bp), 14: pCT5-bac 2.0 cut with BamHI/BsaI (3481bp, 2087bp, 2052bp, 257bp), 15: SDM1 cut with BamHI/BsaI (3481bp, 2309bp, 2087bp), 16: SDM1,3 cut with BamHI/BsaI (5790bp, 2087bp), 17: pCT5c cut with BamHI/BsaI, 18: HyperLadderTM 1kb.


Conclusion

This part has high efficiency and quality in site directed mutagenesis.


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]


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Categories
Parameters
None