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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 [http://partsregistry.org/Part:BBa_B0010 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 [http://partsregistry.org/Part:BBa_B0010 BBa_B0010] (plasmid chosen containing the terminator).
5. Two more iGEM parts have been transformed:
| Part | Characteristic | Resistance | Well (Kit Plate) |
|---|---|---|---|
|
[http://partsregistry.org/Part:BBa_I6007 BBa_I6007] |
Double repressor: called Inverter TetR |
A |
1C (2) |
|
[http://partsregistry.org/Part:BBa_P1010 BBa_P1010] |
Death Cassette |
C |
5E (1) |
Remarks: [http://partsregistry.org/Part:BBa_P1010 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).
