Difference between revisions of "Part:BBa K3407025:Design"
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1rst PCR: | 1rst PCR: | ||
| − | forward primer 5’ - tttataacctccttagagctcga - 3’ | + | forward primer 5’ - tttataacctccttagagctcga - 3’ |
| − | reverse primer 5’ - <b>tgcatccactaaagttaccg </b>gttttagagctagaaatagcaag - 3’ | + | reverse primer 5’ - <b>tgcatccactaaagttaccg </b>gttttagagctagaaatagcaag - 3’ |
Second PCR: | Second PCR: | ||
forward primer 5’- ccaattgtccatattgcatca 3’ | forward primer 5’- ccaattgtccatattgcatca 3’ | ||
| − | reverse primer 5’ - <b>cggtaactttagtggatgcag</b>gtgctcagtatctctatcactga - 3’. | + | reverse primer 5’ - <b>cggtaactttagtggatgcag</b>gtgctcagtatctctatcactga - 3’. |
The sequence of the protospacer is highlighted in bold. | The sequence of the protospacer is highlighted in bold. | ||
A Gibson Assembly was done with both PCR fragments and the resulting plasmid was transformed in <i>E. coli</i> DH5alpha and recovered on LB with 60 ng/µl spectinomycin. The plasmid was confirmed by sequencing. | A Gibson Assembly was done with both PCR fragments and the resulting plasmid was transformed in <i>E. coli</i> DH5alpha and recovered on LB with 60 ng/µl spectinomycin. The plasmid was confirmed by sequencing. | ||
Revision as of 21:23, 27 October 2020
pKDsgRNA_4.3 sgRNA targetting phage T7 gene 4.3
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]
Design Notes
The biobrick is constructed by two PCR’s on a commercially bought plasmid pKDsgRNA-p15 and a Gibson Assembly. The primers used were the following:
1rst PCR:
forward primer 5’ - tttataacctccttagagctcga - 3’ reverse primer 5’ - tgcatccactaaagttaccg gttttagagctagaaatagcaag - 3’
Second PCR:
forward primer 5’- ccaattgtccatattgcatca 3’ reverse primer 5’ - cggtaactttagtggatgcaggtgctcagtatctctatcactga - 3’.
The sequence of the protospacer is highlighted in bold.
A Gibson Assembly was done with both PCR fragments and the resulting plasmid was transformed in E. coli DH5alpha and recovered on LB with 60 ng/µl spectinomycin. The plasmid was confirmed by sequencing.