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Team:The Agency Escondido/notebook
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<tr><td>Grant Hassinger</td><td>100mL LB broth</td><td></td></tr> | <tr><td>Grant Hassinger</td><td>100mL LB broth</td><td></td></tr> | ||
</table> | </table> | ||
| + | <h2>April 16</h2> | ||
| + | <p>Our refrigerated centrifuge arrived, removing the final obstacle to 3A assembly. However, the centrifuge did not consistently power up. This issue was initially ascribed to a faulty fuse.</p> | ||
<h2>April 17</h2> | <h2>April 17</h2> | ||
| - | <p>Andrew Buss prepared 100 mL of LB broth using <a href="http://casp.agency-stem.org/media/G13-LP0003-14.pdf">revision 14 of our LB broth protocol</a>.</p> | + | |
| + | <p>Upon closer inspection of the centrifuge, it became evident that it was in fact missing a fuse. When a 20A fuse was installed, the centrifuge powered up normally. Several test runs indicated temperature control in the range of -10 to 30 degrees C, and an effective maximum centrifuge speed of 2500 RPM (despite a dial that can be turned to 6,000 RPM). </p> | ||
| + | |||
| + | <p>Andrew Buss prepared 100 mL of LB broth using <a href="http://casp.agency-stem.org/media/G13-LP0003-14.pdf">revision 14 of our LB broth protocol</a>. However, autoclaving was deferred until a larger batch of material needed autoclaving.</p> | ||
<p>Andrew Buss inoculated two tubes of LB broth with E. coli containing Part A and Part B from the 4/10 plates to provide additional miniprep material for the following day. Due to the scarcity of amp/kan media (antibiotics to be ordered soon), these were cultured without antibiotics. </p> | <p>Andrew Buss inoculated two tubes of LB broth with E. coli containing Part A and Part B from the 4/10 plates to provide additional miniprep material for the following day. Due to the scarcity of amp/kan media (antibiotics to be ordered soon), these were cultured without antibiotics. </p> | ||
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<p>The tubes containing Part A and Part B had not displayed visible growth from the night before. However, the tubes inoculated on 4/12 displayed growth.</p> | <p>The tubes containing Part A and Part B had not displayed visible growth from the night before. However, the tubes inoculated on 4/12 displayed growth.</p> | ||
| - | + | ||
| - | <p>Andrew Buss extracted plasmid DNA containing Part A and Part B from the cells in the 4/12 tubes using the 3A assembly kit miniprep protocol. RNAse 1 was accidentally omitted from the P1 buffer, so it will need to be introduced before transformation. The two resulting samples were placed in the freezer, labeled "ATB A" and "ATB B". Before restriction can proceed, the concentration of DNA in the samples must be | + | <p>Andrew Buss extracted plasmid DNA containing Part A and Part B from the cells in the 4/12 tubes using the 3A assembly kit miniprep protocol. RNAse 1 was accidentally omitted from the P1 buffer, so it will need to be introduced before transformation. The two resulting samples were placed in the freezer, labeled "ATB A" and "ATB B". Halfway through the process, the non-refrigerated microcentrifuge was relocated to the interior of our refrigerator, because its operation generates enough heat to risk damaging the DNA samples.</p> |
| + | |||
| + | <p>Before restriction can proceed with any of our DNA fragments, the concentration of DNA in the samples must be measured. Our lab does not own a nanodrop machine, so Andrew Segina will use one at a local university to perform the measurement.</p> | ||
<p>Andrew Buss inoculated three LB tubes and one LB plate with NEB10 cells from the 4/10 plate in preparation for transformation the following day.</p> | <p>Andrew Buss inoculated three LB tubes and one LB plate with NEB10 cells from the 4/10 plate in preparation for transformation the following day.</p> | ||
Revision as of 06:06, 19 April 2013
April 9
Andrew Buss prepared 120 mL of LB broth
April 10
Andrew Buss prepared two LB plates without antibiotics, using our LB plate protocol, rev 72.
Andrew Segina inoculated two ampicillin/kanamycin LB plates with E. coli containing Parts A and B from the 3A assembly kit
Andrew Buss inoculated an SOB plate with NEB10 competent E. coli
April 11
Andrew Segina inoculated an SOB plate with NEB10 competent E. coli
The plates previously inoculated with E. coli with parts A and B do not show visible growth
Andrew Segina prepared 1 L of YPD broth without dextrose
April 12
Andrew Buss inspected plates at 8:15 AM. Both NEB10 plates showed visible growth. Neither plate with Part A or B displayed growth.
Andrew Buss prepared four YPD plates from the 1L stock and mixed 4 grams of table sugar before pouring
Andrew Buss inoculated two LB tubes each with E. coli with parts A and B directly from the agar stabs included in the 3A assembly kit. The intention was to diagnose the lack of growth on the A and B plates over two days.
Andrew Buss inspected plates again around 1:00 PM. NEB10 plates showed additional growth, and the plate with Part A contained visible colonies. The plate with Part B did not show growth, however.
Grant Hassinger mixed 100 mL of LB broth and began an autoclave cycle with the media
Vansh Singh inoculated a tube of ampicillin/kanamycin LB broth from the 3A assembly kit with a colony from the Part A plate
Vansh Singh inspected Part B plate at 10:20pm. Part B plate showed visible growth.
Vansh Singh inoculated a tube of ampicillin/kanamycin LB broth from the 3A assembly kit with a colony from the Part B plate
Vansh Singh prepared a tube of ampicillin/kanamycin LB broth from the 3A assembly kit without inoculation. The tube was placed into the refrigerator
Andrew Buss inoculated a tube of LB broth with a colony from the NEB10 plate from April 10
April 13 labeled media
| Andrew Segina | LB plate | Part A from kit | Ampicillin, kanamycin | |
| Andrew Segina | LB plate | Part B from kit | Ampicillin, kanamycin | |
| Andrew Buss | SOB plate | NEB 10 from kit | ||
| Andrew Segina | SOB plate | NEB 10 from kit | ||
| Andrew Buss | LB tube | Part A from kit | Incubating in dry bath | |
| Andrew Buss | LB tube x2 | Part A from kit | Incubating in dry bath | |
| Andrew Buss | LB tube x2 | Part B from kit | Incubating in dry bath | |
| Vansh Singh | LB tube | Part A from 4/10 plate | Incubating in dry bath | |
| Vansh Singh | LB tube | Part B from 4/10 plate | Incubating in dry bath | |
| Andrew Buss | LB tube | NEB 10 from 4/10 SOB plate | Incubating in dry bath |
April 13 available media
| Andrew Segina | 800 mL YPD | Lacks dextrose |
| Andrew Buss | 4 YPD plates | |
| Andrew Segina | 8 LB plates | |
| Andrew Buss | ~10mL LB tube | In dry bath for comparison |
| Andrew Buss | ~40mL LB broth | |
| Vansh Singh | ~5mL LB broth | Ampicillin, kanamycin |
| Grant Hassinger | 100mL LB broth | |
| Grant Hassinger | 100mL LB broth |
April 16
Our refrigerated centrifuge arrived, removing the final obstacle to 3A assembly. However, the centrifuge did not consistently power up. This issue was initially ascribed to a faulty fuse.
April 17
Upon closer inspection of the centrifuge, it became evident that it was in fact missing a fuse. When a 20A fuse was installed, the centrifuge powered up normally. Several test runs indicated temperature control in the range of -10 to 30 degrees C, and an effective maximum centrifuge speed of 2500 RPM (despite a dial that can be turned to 6,000 RPM).
Andrew Buss prepared 100 mL of LB broth using revision 14 of our LB broth protocol. However, autoclaving was deferred until a larger batch of material needed autoclaving.
Andrew Buss inoculated two tubes of LB broth with E. coli containing Part A and Part B from the 4/10 plates to provide additional miniprep material for the following day. Due to the scarcity of amp/kan media (antibiotics to be ordered soon), these were cultured without antibiotics.
Andrew BussApril 18
The tubes containing Part A and Part B had not displayed visible growth from the night before. However, the tubes inoculated on 4/12 displayed growth.
Andrew Buss extracted plasmid DNA containing Part A and Part B from the cells in the 4/12 tubes using the 3A assembly kit miniprep protocol. RNAse 1 was accidentally omitted from the P1 buffer, so it will need to be introduced before transformation. The two resulting samples were placed in the freezer, labeled "ATB A" and "ATB B". Halfway through the process, the non-refrigerated microcentrifuge was relocated to the interior of our refrigerator, because its operation generates enough heat to risk damaging the DNA samples.
Before restriction can proceed with any of our DNA fragments, the concentration of DNA in the samples must be measured. Our lab does not own a nanodrop machine, so Andrew Segina will use one at a local university to perform the measurement.
Andrew Buss inoculated three LB tubes and one LB plate with NEB10 cells from the 4/10 plate in preparation for transformation the following day.
