Difference between revisions of "Part:BBa K2030001:Design"
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===Design Notes=== | ===Design Notes=== | ||
| − | The | + | The sequence of <i>Saccharomyces cerevisiae</i> [http://www.yeastgenome.org/contig/JRIV01000108.1/overview CEN.PK] chromosome XI was used to design primers for amplification of this promoter. |
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| − | The primers were designed to anneal at 63 | + | The primers were designed to anneal at 63 °C (although PCR worked better at 61 °C), using Phusion High-Fidelity DNA Polymerase. |
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| − | For assembly of this biobrick, the vector pSB1C3 (BBa_J04450) was cut with XbaI and PstI and purified from gel. The PCR product of pPCK1 was cut with XbaI and PstI and spin column purified. The two pieces was ligated and chemically transformed into E. coli | + | For assembly of this biobrick, the vector pSB1C3 ([https://parts.igem.org/Part:BBa_J04450 BBa_J04450]) was cut with XbaI and PstI and the 2044 bp band purified from gel. The PCR product of pPCK1 was cut with XbaI and PstI and spin column purified. The two pieces was ligated and chemically transformed into <i>E. coli</i> DH5α and plated on LB + chloramphenicol. False positive colonies still contain the RFP from BBa_J04450 and are red. White colonies were restreaked on LB + chloramphenicol and their plasmids extracted using miniprep. The plasmids were verified by digestion with XbaI and PstI, resulting in two bands: 2044 (vector) and 797 (pPCK1). The biobrick was then sent for sequencing for further verification. |
===Source=== | ===Source=== | ||
| − | Genomic DNA from Saccharomyces cerevisiae CEN.PK113-5D. | + | Genomic DNA from <i>Saccharomyces cerevisiae</i> CEN.PK113-5D. |
===References=== | ===References=== | ||
Latest revision as of 11:29, 9 October 2016
pPCK1 S. cerevisiae promoter
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 221
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 364
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The sequence of Saccharomyces cerevisiae [http://www.yeastgenome.org/contig/JRIV01000108.1/overview CEN.PK] chromosome XI was used to design primers for amplification of this promoter.
FW: gccgcttctagagTCGTTCGTTGTACGTACATTTAC
RV: ttcttcctgcagcggccgctactagtaGTTGTTATTTTATTATGGAATAATTAGTTGC
The primers were designed to anneal at 63 °C (although PCR worked better at 61 °C), using Phusion High-Fidelity DNA Polymerase.
The uppercase nucleotides hybridizes with the template and the lowercase nucleotides contains the overhang. The overhang in the FW primer contains the last 13 bp of the prefix, which includes 6 protective bases before the XbaI site to ensure efficient cutting. The overhang in the RV primer contains the suffix with additional 6 protective bases to ensure efficient cutting with PstI.
For assembly of this biobrick, the vector pSB1C3 (BBa_J04450) was cut with XbaI and PstI and the 2044 bp band purified from gel. The PCR product of pPCK1 was cut with XbaI and PstI and spin column purified. The two pieces was ligated and chemically transformed into E. coli DH5α and plated on LB + chloramphenicol. False positive colonies still contain the RFP from BBa_J04450 and are red. White colonies were restreaked on LB + chloramphenicol and their plasmids extracted using miniprep. The plasmids were verified by digestion with XbaI and PstI, resulting in two bands: 2044 (vector) and 797 (pPCK1). The biobrick was then sent for sequencing for further verification.
Source
Genomic DNA from Saccharomyces cerevisiae CEN.PK113-5D.