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TUDelft/22 August 2008
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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. | 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. | ||
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Revision as of 16:08, 25 August 2008
22th August
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 due to the soaking of the water in the punch, reducing free liquid greatly. 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 (also blanc) | 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 = 42 | 11.04 | 0.75 |
| 10 ul TE + 1002 9A punch, T = 42 + 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 |
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.
