1155 pET28b-Ara-S1
Cloning of Arabidopsis S1 Nuclease into 1155 pET28b
[edit]Purpose: Cloning of Arabidopsis S1 Nuclease into 1155 pET28b
Date: 06/05/2012
Notebook:
Page: 008803-003308
Clone ID: 1155
Experimenters: Jennifer Truong
Cloning
[edit]See 1154 (mistake of name, using a different plasmid)
6/5/2012: 1154-5 sequencing came back mutated, restarted experiment from the beginning:
Synthesized PCR product with PHUSION enzyme, ran PCR gel, cut out product for purifaction
Digested 1155 and pET28b (vector) with Nco1 + Xho1, cut out bands, ligated and transformed cells with DH5 alpha and grew on KAN plates
06/06/12: Nothing grew on the KAN plates
Cut out the wrong band from the day before (see above), redid gene synthesis and cut out circled band
Digested purified DNA and pET28b (V12) vector with Nco1 + Xho1, cut out bands for ligation
Redid gene synthesis, cut out band, purified for digestion
Digested purified DNA and pET28b (V12) vector with Nco1 + Xho1, cut out bands for ligation
06/07/2012: Again, no growth on plates.
Synthesized PCR product again, cut out circled band, purified DNA, digested DNA and vector for digestion
Digested purified DNA and pET28b (V12) vector with Nco1 + Xho1, cut out bands for ligation (lanes 1 and 2)
Colony Screening
[edit]Date: 06/08/2012
All screening were positive, grew samples 1-6 in media (lanes 1-6)
Performed miniprep on samples, digested with enzymes to check for inserts, submitted sample #2 (lane 8) for sequencing.
Result intepretation/Summary
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Conclusion
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