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TUDelft/22 August 2008
From 2008.igem.org
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August 22nd
DNA extraction test
Today another gel was run with DNA soaked from the spots. To test the soaking-protocol, various steps of the protocol were performed on the DNA. This time 10 ul of TE was used, because due to the soaking of the water into the punch, using 5 ul reduced free liquid to be added to cells (or for analysis) below 2 ul. Again various conditions were tested, and also the later sent spots from the promoter strength testing DNA were analyzed. To give an idea of DNA concentrations, all spots were nanodropped. These results are in the table below. For this testing we used a spot we won't be using and showed a good band on the gel of the repository. 1002 9A, a lock from Berkeley06, which had inconsistent sequence.
| Sample | ng/ul | A260/A280 |
|---|---|---|
| TE (blank) | 0.53 | 1.42 |
| 10 ul TE + 1002 9A punch, Tr | 4.94 | 0.69 |
| 10 ul TE + 1002 9A punch, Tr + spinned | 9.69 | 0.89 |
| 10 ul TE + 1002 9A punch, T = 42ºC | 11.04 | 0.75 |
| 10 ul TE + 1002 9A punch, T = 42ºC + Spinned | 11.78 | 1.23 |
| 10 ul TE + promoter strength punch, Tr | 5.94 | 0.97 |
| 10 ul TE + promoter strength punch, 42 C + spinned | 4.81 | 1.36 |
| 10 ul TE + 3 ul pSB1A7 (isolated on 20-08-08) | 11.69 | 1.66 |
Tr = Room Temperature
These results have again rather low 260/280, but DNA concentrations that should be visible on a gel. Concentrations this time with increased following of iGEM procedure.
The gel showed no visible DNA bands for the soaked punches. The positive control did show bands, as can be seen below.
Transformation
A transformation has been started today for testing of the midiprepped vectors. To prepare for the arrival of our thermosensitive sequences next week we also tried to transform a couple of parts for our constructs, although if these fail we will need a different way to obtain the parts. The parts (apart from the midiprepped) used for a transformation are:
| Part | ng/ul | A260/A280 |
|---|---|---|
| I712019 | 14.22 | 0.64 |
| B0015 | 14.48 | 0.68 |
| I13453 | 10.16 | 0.88 |
| R0010 | 4.44 | 0.66 |
| B0030 | 11.33 | 0.67 |
| I742152 | 20.2 | 0.69 |
Punches were no longer obtained by the punch tool but by cutting them out with a scalpel, due to time gain and the punch tool not working very well. All these punches were soaked in 10 ul TE and transformations were started with 5 ul of extracted DNA. After transformation cells were plated on LB+Amp.
