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Team:TU Delft/21 July 2010 content
From 2010.igem.org
Contents |
Lab work
Ordered DNA
The transformations of 19 July containing the different ligations gave colonies (~5 per plate). We picked 5 colonies per plate and performed a colony PCR.
Alkane degradation
Unfortunately there were no transformants on yesterday's plates. This is most likely due to the fact that the ligation didn't work with the small pieces of DNA of the RBSs. Next plan is to cut open the RBS plasmid without removing the RBS (with SpeI and PstI) and insert the gene in this plasmid. We will try this tomorrow.
Salt Tolerance
The overnight ligation was transformed using the standard method and grown up overnight at 37 degrees C.
Emulsifier
Lane Description
| # | Description | Expected lenght (bp) | Primer | Status |
| M1 | EZ Ladder | n/a | n/a | n/a |
| 1 | B0032 (control) | 220 | G00100 + G00101 | ✓ |
| 2 | Transformant #1 of ligation mix R0011-B0032 | 300 | G00100 + G00101 | ✗ |
| 3 | Transformant #2 of ligation mix R0011-B0032 | 300 | G00100 + G00101 | ✗ |
| 4 | Transformant #3 of ligation mix R0011-B0032 | 300 | G00100 + G00101 | ✗ |
| 5 | Transformant #4 of ligation mix R0011-B0032 | 300 | G00100 + G00101 | ✗ |
| 6 | Transformant #5 of ligation mix R0011-B0032 | 300 | G00100 + G00101 | ✗ |
| 7 | Transformant #6 of ligation mix R0011-B0032 | 300 | G00100 + G00101 | ✗ |
| 8 | Transformant #7 of ligation mix R0011-B0032 | 300 | G00100 + G00101 | ✗ |
Unfortunately all other lanes were empty or the same as the negative control. So from this gel we conclude that the ligation has failed.
Characterisation of Anderson RBS sequences
Assembly of reference construct
Method 1
Hypothesis II The transformants that were replated on kanamycin plates produced colonies, indicating that hypothesis II was invalid.
Method 4
The obtained colonies on ampicillin plates were screened for GFP fluorescence, those that fluoresced were inoculated in 5 mL of LB medium for over night growth.
