EPF-Lausanne/9 July 2009

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===Wet Lab===
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==Wet Lab==
1. Miniprep and isolations of the yesterday transformed plasmids. (cf. [https://2009.igem.org/Team:EPF-Lausanne/Notebook/Wet_Lab#08.07.09 08.09.09 subpart])
1. Miniprep and isolations of the yesterday transformed plasmids. (cf. [https://2009.igem.org/Team:EPF-Lausanne/Notebook/Wet_Lab#08.07.09 08.09.09 subpart])
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==Cloning Strategy==
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===Cloning Strategy===
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'''Partial digestion strategy'''
'''Partial digestion strategy'''
:* Problem to overcome:  
:* Problem to overcome:  
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:Most concentrated tube: all P sites are recognized and cut, so the number of parts diminishes with the concentration value.
: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).
:Run an agarose gel and extract the right piece (recognized by the segment's length).
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==People in the lab==
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: Heidi, Tu, Nath, Rafael, Basile
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Latest revision as of 09:12, 28 July 2009



Contents


9 July 2009



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:

Parts&Characteristics
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).


People in the lab

Heidi, Tu, Nath, Rafael, Basile



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