Difference between revisions of "Team:LambertGA/Notebook"
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Revision as of 02:43, 16 October 2016
Notebook
Safety procedures and aseptic technique was reviewed by our iGEM supervisor: Janet Standeven.
This week was subjected to ligating two of our three parts of the genetic construct together: p-lambda-r/Lac I/ tspurple/ deg. Tag (LAA or DAS tags) + ROO1/ ClpXP/ CI
Note: no construct was ligated without a present degradation tag
After ligation was complete the the genetic constructs were then transformed into separate DH10 E. coli cells
The previous transformation and ligation was unsuccessful due to the lack of ribosomal binding sites in the genetic constructs
We were required to re-digest, re-transform and re-ligate the p-lambda-r/ LacI/ TsPurple/ deg.tag(LAA or DAS) + ROO11/ ClpXP/ CI
Transformation and ligation of the third part: genetic construct without a degradation tag present
The continuation of the re-ligations and re-transformations during week 3 were ultimately unsuccessful due to RFP( red fluorescent protein) contamination
Miniprep and nanodrop of the genetic construct without a present degradation tag in the backbone: 1C3.
Further detection of previous errors
Liquid culture of genetic construct without present degradation tag
Inoculation of RFP and previous TS purple colonies
Due to several unsuccessful procedures, we decide to religate p-lamba-r/LacI + ts purple (deg tag) and R0011/ClpXP + CI
The above ligations were also transformed
Successful transformation of TsPurple and GFP constructs with variation of degradations tag (LAA, DAS and no degradation tag) and IPTG levels ( 1 uM, 10 uM, 100 Um, 1 mM)
Ligation
