![](https://parts.igem.org/images/partbypart/icon_primer.png)
Primer
SDM3_Rv
Part:BBa_K4417006
Designed by: Jiaying Zou Group: iGEM22_UCL (2022-09-29)
SDM3 Reverse Primer for pCT5c Mutation
Description
This primer is designed for mutating the BsaI site 3 in pCT5-bac 2.0. This part is a reverse primer with a mutation site, changing t to c.
Usage and Biology
- This part is used in site directed mutagenesis of pCT5c (Part: BBa_K4417000).
- Sequence: gaaaagtgaggccatggagag
- Length: 21-mer
- Tm: 51℃
- GC content: 52%
Method
This part is used with BBa_K4417000 and BBa_K4417005. A detailed protocol is described in BBa_K4417000.
Characterization
After two rounds of SDM, this primer will remove the final BsaI site, giving a Type IIS compatible plasmid. 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 5, 9, 13, and 17.
![](/wiki/images/3/31/Zjy13.png)
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:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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Categories
Parameters
None |