Team:SDU-Denmark/labnotes5
From 2010.igem.org
(Difference between revisions)
(→Lab notes (8/9 - 8/15)) |
(→Lab notes (8/9 - 8/15)) |
||
Line 101: | Line 101: | ||
<br><br> | <br><br> | ||
Notes:<br> | Notes:<br> | ||
- | Over night cultures | + | Over night (ON) cultures were grown from 4 colonies, until the following OD’s were obtained: |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | = | + | Top 10 - no insert OD = 0,008 (100 x diluted)<br> |
- | + | Top 10 - with K274210 insert OD = 0,011 (100 x diluted)<br> | |
- | + | MG1655 - no insert OD = 0,020 (100 x diluted)<br> | |
- | + | Mg1655 - with K274210 insert OD = 0,017 (100 x diluted)<br> | |
- | + | ||
- | <br> | + | ON cultures were grown in 110 ml LB media. Colonies with K274210 insert were grown in LB media containing ampicillin. |
- | + | All ON cultures were grown for 20 hours at 37 °C. | |
- | 100 mL | + | After 16 hours, 10 ml of the ON cultures were transferred into 110 ml LB media and grown for 4 hours to reach the exponential phase, where the following OD’s were obtained: |
- | A | + | |
- | The samples | + | Top 10 - no insert OD = 0,049 (100 x diluted)<br> |
- | + | Top 10 - with K274210 insert OD = 0,044 (100 x diluted)<br> | |
+ | MG1655 - no insert OD = 0,007 (100 x diluted)<br> | ||
+ | Mg1655 - with K274210 insert OD = 0,009 (100 x diluted)<br> | ||
+ | |||
+ | Cultures with K274210 insert were grown in media containing ampicillin. | ||
+ | 100 mL cell culture were centrifuged for 5 min at 14000 RPM. The supernatant was discarded and cells were resuspended in 5 mL acetone (99,9%), except the Top 10 E. coli with the K274210 insert, which was resuspended in 10 mL acetone (source of error). The resuspended cells were sonicated for 5 min. Samples were spun down, the supernatant was transferred to new tubes, and cell debris was discarded. A standard curve was made from pure beta-carotene.<br> | ||
+ | |||
+ | The samples at the OD’s seen above as well as solutions of pure beta-carotene with known concentrations were measured at a fixed wavelength of 456 nm. | ||
+ | Known concentrations and their absorbances: | ||
<table style="text-align: left; width: 100%;" border="1" | <table style="text-align: left; width: 100%;" border="1" | ||
cellpadding="2" cellspacing="2"> | cellpadding="2" cellspacing="2"> | ||
Line 177: | Line 173: | ||
</tr> | </tr> | ||
</table> | </table> | ||
- | The samples:<br> | + | The samples and their absorbances:<br> |
<table style="text-align: left; width: 100%;" border="1" | <table style="text-align: left; width: 100%;" border="1" | ||
cellpadding="2" cellspacing="2"> | cellpadding="2" cellspacing="2"> | ||
Line 206: | Line 202: | ||
</tr> | </tr> | ||
</table> | </table> | ||
- | + | UV-VIS absorbance spectra of the known solutions were obtained, as well as spectra of the samples at the ODs shown above. The spectra of Top 10 and MG1655 in the stationary phase as well as selected spectra of known solutions are shown below:<br> | |
[[Image:Team-SDU-denmarkBeta-carotene.jpg |400px]] | [[Image:Team-SDU-denmarkBeta-carotene.jpg |400px]] | ||
=== PCR of NinaB (again) === | === PCR of NinaB (again) === |
Revision as of 06:19, 31 August 2010