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EPF-Lausanne/9 July 2009
From 2009.igem.org
Wet Lab
1. Miniprep and isolations of the yesterday transformed plasmids. (cf. 08.09.09 subpart)
Concentrations of the plasmids: cf. lab notebook pp. 8-9
2. PCR of pCal-n to PCR up LOVTAP with the different primers designed the 06.09.09, the products are:
- Prom_T7-RBS-CBP-LOVTAP
- RBS-CBP-LOVTAP
- CBP-LOVTAP
- LOVTAP
Result: Prom_T7-RBS-CBP-LOVTAP didn't worked, the other were ok.
3. An agarose gel was runned to check PCR products
4. PCR products were digested with EcorI and SpeI and BBa_B0010 (plasmid chosen containing the terminator) was digested with EcorI and XbaI, the digestion products were treated with phosphatase. Then, PCR products were purified.
Finally, LOVTAP (PCR products) were ligated on BBa_B0010 (plasmid chosen containing the terminator).
5. Two more iGEM parts have been transformed:
| Part | Characteristic | Resistance | Well (Kit Plate) |
|---|---|---|---|
|
Double repressor: called Inverter TetR |
A |
1C (2) | |
|
Death Cassette |
C |
5E (1) |
Remarks: BBa_P1010, the death gene has to be transformed in ccdB (death gene) resistant cells: One Shot ccdB survival 2T1 E. Coli
Cloning Strategy
Partial digestion strategy
- Problem to overcome:
- PstI sites in LOVTAP sequence -> Impossible to use iGEM protocol for ligation (where downstream part must be cut with XP)
- Strategy:
- Cut with X first.
- Divide into several solutions and add different concentrations of P (by dilution series)
- Results:
- Most concentrated tube: all P sites are recognized and cut, so the number of parts diminishes with the concentration value.
- Run an agarose gel and extract the right piece (recognized by the segment's length).
People in the lab
- Heidi, Tu, Nath, Rafael, Basile
