Difference between revisions of "Team:Tufts/Results"
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− | In addition to the creation of the Cas9-aTcdB protein, the lab also worked on the creation of a template for the easy customization of the gRNA for T7 driven transcription. This construct was obtained as a gBlock, amplified, and inserted into the pSB1C3 vector with restriction cloning using EcoRI and PstI. Colonies lacking RFP were used to inoculate liquid cultures, whose plasmids were ultimately purified and sequenced. Sequencing confirmed successful insertion. This construct now constitutes our Biobrick part BBa_K1818000. | + | In addition to the creation of the Cas9-aTcdB protein, the lab also worked on the creation of a template for the easy customization of the gRNA for T7 driven transcription. This construct was obtained as a gBlock, amplified, and inserted into the pSB1C3 vector with restriction cloning using EcoRI and PstI. |
+ | <center><img src = "https://static.igem.org/mediawiki/2015/f/f1/Tuftsplate.jpg" height="30%" width="30%"/></center> | ||
+ | |||
+ | Colonies lacking RFP were used to inoculate liquid cultures, whose plasmids were ultimately purified and sequenced. Sequencing confirmed successful insertion. This construct now constitutes our Biobrick part BBa_K1818000. | ||
Unfortunately, an attempt to insert a pair of phosphorylated annealed oligos into this template was unsuccessful, as confirmed by sequencing which shows that the BbsI did not cut the vector or that the vector re-ligated. | Unfortunately, an attempt to insert a pair of phosphorylated annealed oligos into this template was unsuccessful, as confirmed by sequencing which shows that the BbsI did not cut the vector or that the vector re-ligated. |
Latest revision as of 03:32, 19 September 2015
The first few steps of the project involved purifying and checking both of the genes for Cas9 and aTcdB. This involved purifying the products of PCR and running them on agarose gels, as well as using a spectrophotometer to identify the concentration and purity of DNA recovered.
1. pHis1522-aTcdBSample | Concentration (ng/μL) |
A | 135.5 |
B | 170.2 |
Sample | Concentration (ng/μL) |
A | 32.9 |
B | 33.3 |
C | 118.3 |
Sample | Concentration (ng/μL) |
A | 45.7 |
B | 46.3 |
C | 40.4 |
Sample | Concentration (ng/μL) |
1 | 49.9 |
2 | 72.1 |
3 | 1702.6 |
4 | 426.6 |
5 | 42.7 |