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Team:Bonn/Notebook
From 2012.igem.org
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===== 06.06 - 06.08 ===== | ===== 06.06 - 06.08 ===== | ||
| - | First restrictions, some of them were successful. The failure on our | + | 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 ===== | ===== 08.06 - 03.07 ===== | ||
| - | PCR, transformation and restriction troubleshooting to find the issue in our | + | 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 in with other biobricks and sample plasmids. So we finally send the LovTAP iGEM plasmid to a company for sequencing. |
| Line 52: | Line 52: | ||
===== 04.07 ===== | ===== 04.07 ===== | ||
Sequencing results of all our biobricks are available: Sequence is okay for RBS32, LacZα, pLac and our double-terminator. Frameshift found in lacI plasmid. | Sequencing results of all our biobricks are available: Sequence is okay for RBS32, LacZα, pLac and our double-terminator. Frameshift found in lacI plasmid. | ||
| - | + | LovTAP was garbage. | |
===== later this month... ===== | ===== later this month... ===== | ||
| Line 64: | Line 64: | ||
* Restrictions | * Restrictions | ||
* Ligations | * Ligations | ||
| + | |||
| + | Midi-Preparation of LovTAP-pCal-n (wt) - we later used this construct for testing reasons only. | ||
===== 07.08 ===== | ===== 07.08 ===== | ||
| Line 69: | Line 71: | ||
===== 08.08 ===== | ===== 08.08 ===== | ||
| - | Restriction analysis of constructs LacZα-TT-pSB1C3 and ccdB-TT-pSB1C3 | + | * Restriction analysis of constructs LacZα-TT-pSB1C3 and ccdB-TT-pSB1C3 |
| + | * Started work on constructs J23119-lacI-pSB1C3 and pLac-RBS32-pSB1K3 | ||
===== 10.08 ===== | ===== 10.08 ===== | ||
| - | Sequencing of | + | * 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 ===== | ||
| + | PCR of modLOV | ||
| + | |||
| + | ===== 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 use in pLac-RBS32-LOV-pSB1C3 construct. | ||
| + | ===== 23.08-28.08 ===== | ||
| + | Creation/finalization of constructs J23119-LacI-pSB1C3 and J23119-LacI-pSBA3 | ||
==== September 2012 ==== | ==== September 2012 ==== | ||
| - | + | ===== 5.09-7.09 ===== | |
| + | Formation of construct pLac-RBS32-LOV-pSB1C3 | ||
=== Protocols === | === Protocols === | ||
:: TODO | :: TODO | ||
(Protocols will have to be '''linked''' here, please don't add the big texts directly!) | (Protocols will have to be '''linked''' here, please don't add the big texts directly!) | ||
Revision as of 21:00, 17 September 2012
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Contents |
Lab Notebook
Everything we did in our lab, including the standard protocols we used.
Summary
- TODO
Highlights by date
May 2012
23.05
SOC medium and SOB medium prepared, microbial culture: DH5α Agar plates, chemo-competent DH5α
31.05
First successful transformation of iGEM plasmids. XL1-blue bacteria was much more competent than our DH5α, so we used these for now.
June 2012
04.06
First Midi-Preps of iGEM plasmids from our distribution kit.
06.06 - 06.08
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 in 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. Frameshift found in lacI plasmid. LovTAP was garbage.
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
PCR of modLOV
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 use in pLac-RBS32-LOV-pSB1C3 construct.
23.08-28.08
Creation/finalization of constructs J23119-LacI-pSB1C3 and J23119-LacI-pSBA3
September 2012
5.09-7.09
Formation of construct pLac-RBS32-LOV-pSB1C3
Protocols
- TODO
(Protocols will have to be linked here, please don't add the big texts directly!)
