Difference between revisions of "Part:BBa K3407008:Design"
| Line 11: | Line 11: | ||
1rst PCR: | 1rst PCR: | ||
| − | forward primer 5’ | + | 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’. | ||
Latest revision as of 21:24, 27 October 2020
sgRNA targetting 4.3 gene of T7 bacteriophage
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.