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Team:Goettingen/NoteBook w5
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Restriction of parts 1 – 4 to cut out promoters for ligation with operator plasmid and restriction of part 8 for inserting GFP-Terminator cassette
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Reactions according to manual of Distribution kit:<o:p></o:p>
- Parts 1 – 3: restriction of 1 µg plasmid DNA with SpeI and EcoRI-HF; 100 µl in total (DNA, enzyme amounts and buffer amount doubled compared to common 50 µl reaction; but distributed to 2 epis with 50 µl each à more complete reaction)<o:p></o:p>
- Part 4: restriction of ca. 250 ng plasmid DNA (8.7 µl from 29.5 ng/µl solution) with SpeI and EcoRI-HF<o:p></o:p>
- Part 8: restriction of 500 ng plasmid DNA with SpeI and PstI<o:p></o:p>
- For parts 1,2, and 3: MasterMix consisting of both enzymes and buffer and sufficient for 4 reactions was used<o:p></o:p>
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è 20 min at 37 °C digestion<o:p></o:p>
è 20 min at 80 °C heat-kill<o:p></o:p>
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- 1xTAE-1%agarose gel<o:p></o:p>
- Load 3 µl of each digest (ca. 30 ng) and 1 µl of plasmid as a control, supplied with 5xDLD<o:p></o:p>
- 3 µl of log ladder as marker<o:p></o:p>
- Run at 100 V<o:p></o:p>
- EtBr staining + destaining<o:p></o:p>
Gel:<o:p></o:p>
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è Still, R.D. is partial à next time: try 30 min incubation<o:p></o:p>
è One can conclude linearization of all plasmids, but promoter fragments are not visible…. Try higher concentrated gel and load higher amounts of DNA<o:p></o:p>
è Reactions stored at – 20 °C<o:p></o:p>
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Transformation from 27.6.13<o:p></o:p>
- Colonies grew on all plates except for negative controls and those of pSB1C3 transformation<o:p></o:p>
- Colonies on without-insert-control of linearized pSB1C3 look slightly pink à remaining template from PCR transformed = pSB1C3 plasmid?<o:p></o:p>
- Colonies on without-insert-control of linearized part 7 vector might result from not digested plasmid (partial digestion, as usual….)<o:p></o:p>
- Just 1 clone for Riboswitch biobrick C…<o:p></o:p>
- Inoculation of 5 clones for each (except for Ribo C) transformation; for without-insert-controls, 3 clones each were inoculated; 4 ml LB+Cm (35 µg/ml)<o:p></o:p>
- Incubation ON<o:p></o:p>
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New plasmid preparation for parts 1 – 4<o:p></o:p>
- Inoculation of parts 1, 3, 4 C1 and part 2 C2 in 4 ml LB+Amp<o:p></o:p>
- Incubation ON<o:p></o:p>
Re-streak clones for parts 6 and 7 on 35 µg/ml Cm plates + incubation on<o:p></o:p>
Purification of part 8 (RD) products from today<o:p></o:p>
PCR clean-up with Qiagen kit<o:p></o:p>
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Elution with 50 µl HPLC water (pre-warmed)<o:p></o:p>
NanoDrop:<o:p></o:p>
|
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Concentration (ng/µl)<o:p></o:p> |
A260nm/A280nm<o:p></o:p> |
A260nm/A230nm<o:p></o:p> |
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part 8 purification<o:p></o:p> |
7.5<o:p></o:p> |
1.34<o:p></o:p> |
1.39<o:p></o:p> |
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Either just low amounts of DNA in the sample or the purification was not very efficient. Eluded a second time
with 20µl, no DNA found.<o:p></o:p>
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