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From 2012.igem.org

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Lab Notebook

Everything we did in our lab, including the standard protocols we used.

Summary

TODO

Highlights by date

May 2012

23.05

Starting our lab work with preparing some media like SOB and SOC and creating chemo-competent DH5α bacteria.

31.05

First successful transformation of iGEM plasmids (pLac, Lac1, LovTap, J23119, LacZalpha, ccdB, RBS32 and the double terminator). XL1-blue bacteria turned out to be much more competent than our DH5α, so we used these from now on.

June 2012

04.06

Midi-Preps of iGEM plasmids from our distribution kit.

06.06 - 08.06

First restrictions, some of them were successful. The failure on our LovTAP plasmid was misinterpreted: We thought we had problems with the restriction, PCR or our transformation, but in fact the iGEM LOV biobrick was broken.

08.06 - 03.07

PCR, transformation and restriction troubleshooting to find the issue in our LovTAP transformation. All tests and modifications of our protocols failed for the LovTAP plasmid, but worked out with other biobricks and sample plasmids. So we finally send the LovTAP iGEM plasmid to a company for sequencing.

July 2012

04.07

Sequencing results of all our biobricks are available: Sequence is okay for RBS32, LacZα, pLac and our double-terminator. A frameshift was found in lacI plasmid. LovTAP was contaminated with another sequence as the found sequence didn't match the desired one at all.

later this month...

Further troubleshooting and optimization of our standard procedures. We also tried to figure out if only our distribution plate contained the broken LOV, so we asked other iGEM teams to provide us with samples from their plates. We did transformations and sequencing, but these LOV plasmids showed the same pattern of degeneration our LOV did.

August 2012

06.08

First application of the 3A assembly, following the official iGEM protocol. (we used a different one before).

• Restrictions
• Ligations

Midi-Preparation of LovTAP-pCal-n (wt) - we later used this construct for testing reasons only.

07.08

Transformation of constructs prepared the day before.

08.08

• Restriction analysis of constructs LacZα-TT-pSB1C3 and ccdB-TT-pSB1C3
• Started work on constructs J23119-lacI-pSB1C3 and pLac-RBS32-pSB1K3

10.08

• Sequencing of our first constructs (LacZα-TT-pSB1C3 and ccdB-TT-pSB1C3) for verification.
• First successful PCR of wildtype-LOV (test for future experiments)
• Midi-Preps of LovTAP-pCal-n (Mutant) and mazF-pBAD

16.08

Work on constructs pLac-RBS32-Ccdb-TT-pSB1A3 and pLac-RBS32-LacZα-TT-pSB1A3 started

21.08

Successful amplification of mod-LOV

22.08- 28.08

First trial of ligation to form the new constructs pLac-RBS32-LOV-Ccdb-TT-pSB1C3 and pLac-RBS32-LOV-Ccdb-TT-pSB1C3

29.08

Purification of the pSB1C3 fragment, which was cut with EcoRI and SpeI before, for usage in the pLac-RBS32-LOV-pSB1C3 construct.

23.08-28.08

Creation/finalization of constructs J23119-LacI-pSB1C3 and J23119-LacI-pSBA3

September 2012

05.09-07.09

Formation of construct pLac-RBS32-LOV-pSB1C3

06.09

• Successful amplification of MazF fragment
• Constructs MazF-pSB1C3 and MazF-TT-pSB1C3 completed

10.09 - 11.09

Mutagenesis PCR No 1: pLac-LOV-pSB1C3 and LOV-pSB1C3 to make the LOV part BioBrick compliant

13.09 - ????

New constructs:

• pLac-RBS32-LOV-Ccdb-TT-pSB1C3
• pLac-RBS32-LOV-LacZalpha-TT-pSB1C3
• pLac-RBS32-LOV-MazF-TT-pSB1C3

Protocols

TODO

(Protocols will have to be linked here, please don't add the big texts directly!)