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| <ul class="menu"> | | <ul class="menu"> |
| <li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/TdT_project">De Novo Synthesis </a></li> | | <li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/TdT_project">De Novo Synthesis </a></li> |
- | <li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Telomere_project">Programmable Lifespan</a> </li> | + | <li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Telomere_project">Programmable Lifespan Timer</a> </li> |
| <li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Biohack_project">Biohacker Kit </a></li> | | <li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Biohack_project">Biohacker Kit </a></li> |
| <li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Hardware">OpenSource Hardware </a> </li> | | <li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Hardware">OpenSource Hardware </a> </li> |
| + | <!--<li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Modeling">Modeling </a> </li>--> |
| <li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Parts">BioBrick Parts </a></li> | | <li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Parts">BioBrick Parts </a></li> |
- | <!--<li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Modeling">Modeling </a> </li>-->
| |
| </ul> | | </ul> |
| </li> | | </li> |
- | <li class="menu-safety"> <a class="menu" href="https://igem.org/Safety/Safety_Form?team_id=1354">Safety</a> </li> | + | <li class="menu-safety"> Social |
- | <li class="menu-outreach"><a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Outreach">Outreach </a></li>
| + | <ul class="menu"> |
| + | <li class="menu"><a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Safety">Safety</a> </li> |
| + | <li class="menu"><a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Ethics">Ethics</a> </li> |
| + | <li class="menu"><a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Outreach">Outreach </a></li> |
| + | </ul> |
| + | </li> |
| <li class="menu-notebook"> Notebook | | <li class="menu-notebook"> Notebook |
| <ul class="menu"> | | <ul class="menu"> |
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| <li>Checked yeast plates -- all plates grew colonies.</li> | | <li>Checked yeast plates -- all plates grew colonies.</li> |
| <li>Since EST4 knocked out in conjunction with RAD52 kills the yeast, we decided to use EST1 instead of EST4 in the gene deletion for the kill switch project.</li> | | <li>Since EST4 knocked out in conjunction with RAD52 kills the yeast, we decided to use EST1 instead of EST4 in the gene deletion for the kill switch project.</li> |
- | <li>Poured plates -- 3 bottles of amp (100ug/mL). We used 250 μL of 100 mg/mL stock concentration of Ampicillin.</li> | + | <li>Poured plates -- 3 bottles of amp (100μg/mL). We used 250 μL of 100 mg/mL stock concentration of Ampicillin.</li> |
| <li>Poured two 0.8% agarose gels, both about 60 mL in each.</li> | | <li>Poured two 0.8% agarose gels, both about 60 mL in each.</li> |
- | <li>Helped set up Restriction Digest Reaction with <em>De novo</em> group (used General Restriction Enzyme Digest protocol): 3 μL buffer, 3 μL BSA, DNA, H<sub>2</sub>O, and Xho1/Xba1 enzyme.</li> | + | <li>Helped set up Restriction Digest Reaction with <em>De novo</em> group (used General Restriction Enzyme Digest protocol): 3 μL buffer, 3 μL BSA, DNA, H<sub>2</sub>O, and XhoI/XbaI enzyme.</li> |
| </ul> | | </ul> |
| For tomorrow: design check primers for knockout cassettes (programmable kill switch project)<br> | | For tomorrow: design check primers for knockout cassettes (programmable kill switch project)<br> |
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| <ul> | | <ul> |
| <li>Checked if plasmids in the freezer can be used in kill switch project (they can). We have pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP.</li> | | <li>Checked if plasmids in the freezer can be used in kill switch project (they can). We have pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP.</li> |
- | <li>Streaked out plates of pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP, put them in 37 degrees incubator to grow.</li> | + | <li>Streaked out plates of pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP, put them in 37°C incubator to grow.</li> |
| <li>Made glucose 40% with 200g dextrose and distilled water to make 500mL of solution (two 250mL bottles).</li> | | <li>Made glucose 40% with 200g dextrose and distilled water to make 500mL of solution (two 250mL bottles).</li> |
| <li>Made clonat plates: regular YPD plates+clonat</li> | | <li>Made clonat plates: regular YPD plates+clonat</li> |
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| <ul> | | <ul> |
| <li>Researched linearizing the genome for E. coli; found that the method described in EMBO_Cui may not be ideal for the kill switch project.</li> | | <li>Researched linearizing the genome for E. coli; found that the method described in EMBO_Cui may not be ideal for the kill switch project.</li> |
- | <li>Helped <em>De novo</em> group start their double restriction digest.</li> | + | <li>Helped De Novo group start their double restriction digest.</li> |
| <li>Discussed expanding upon yeast system to make it so that the yeast strains programmed with the kill switch cannot mate with wild-type yeast strains.</li> | | <li>Discussed expanding upon yeast system to make it so that the yeast strains programmed with the kill switch cannot mate with wild-type yeast strains.</li> |
| <li>Inoculated the cultures of pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP and left them growing overnight in LB broth with Amp antibiotic.</li> | | <li>Inoculated the cultures of pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP and left them growing overnight in LB broth with Amp antibiotic.</li> |
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| <ul> | | <ul> |
| <li>Checked the cells grown overnight; confirmed the correct plasmids had been grown (pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP)</li> | | <li>Checked the cells grown overnight; confirmed the correct plasmids had been grown (pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP)</li> |
- | <li>Prepared 1mL miniculture of equal part cells and a 60% glycerol stock for each sample to be stored in the -80C freezer. </li> | + | <li>Prepared 1mL miniculture of equal part cells and a 60% glycerol stock for each sample to be stored in the -80°C freezer. </li> |
| <li>miniprepped the rest of the overnight cultures (2 samples of each plasmid), following the Miniprep Protocol with some exceptions. The 5mL of overnight culture was centrifuged at 3900rpm for 10 minutes for the first step of preparing the cell lysate. For the second step of eluting the DNA, 50μl of preheated TE buffer was added to the column instead of the stated 75μl. </li> | | <li>miniprepped the rest of the overnight cultures (2 samples of each plasmid), following the Miniprep Protocol with some exceptions. The 5mL of overnight culture was centrifuged at 3900rpm for 10 minutes for the first step of preparing the cell lysate. For the second step of eluting the DNA, 50μl of preheated TE buffer was added to the column instead of the stated 75μl. </li> |
| <li>After the plasmids were isolated, the concentration of each sample was measured using the nanodrop, then stored at -20°C.</li> | | <li>After the plasmids were isolated, the concentration of each sample was measured using the nanodrop, then stored at -20°C.</li> |
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| <li>Streaked out plasmids onto Amp plates (pAG25, pFA6a-LEU2MX6, pFA6a-TRP1)</li> | | <li>Streaked out plasmids onto Amp plates (pAG25, pFA6a-LEU2MX6, pFA6a-TRP1)</li> |
| <li>Reconstituted primers and made 100x umol stocks of primers with TE buffer</li> | | <li>Reconstituted primers and made 100x umol stocks of primers with TE buffer</li> |
- | <li>Worked with Devora to label and dilute all of the 100 uM conc. of the primers; dilution was 1:10 so the diluted primers should be a 10 umolar concentration (did 10 uL resuspended primer with 90 uL ddH2O). </li> | + | <li>Worked with Devora to label and dilute all of the 100μM conc. of the primers; dilution was 1:10 so the diluted primers should be a 10μM concentration (did 10 uL resuspended primer with 90μL ddH<sub>2</sub>O). </li> |
| <br> | | <br> |
| <dl><dt>List of Primers:</dt> | | <dl><dt>List of Primers:</dt> |
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| <dd><em>De Novo group</em>: PolyA (forward and reverse)</dd></dl> | | <dd><em>De Novo group</em>: PolyA (forward and reverse)</dd></dl> |
| <br> | | <br> |
- | <li>Colony PCR amplified LEU2, TRPI, and pAG25 (we got this by stabbing the colony within the ring in the glass bottles that were ordered and swirling that into 200 uL of ddH2O water) and for each we did 2 sets of PCR; 1 with ESTI deletion primers and the other with RAD52 deletion primers. We did 20 uL of each of the plasmid along with 2.5 uL each of the forward and reverse primers. As a control we ran 20 uL of ddH2O water with 2.5 each of the primers (so a control for both EstI and Rad52). We added an extension time of 2 minutes. We left that to PCR overnight because the machine is heat-regulated so it's fine if it stays in there. </li> | + | <li>Colony PCR amplified LEU2, TRPI, and pAG25 (we got this by stabbing the colony within the ring in the glass bottles that were ordered and swirling that into 200μL of ddH<sub>2</sub>O water) and for each we did 2 sets of PCR; 1 with ESTI deletion primers and the other with RAD52 deletion primers. We did 20μL of each of the plasmid along with 2.5μL each of the forward and reverse primers. As a control we ran 20μL of ddH<sub>2</sub>O water with 2.5 each of the primers (so a control for both EstI and Rad52). We added an extension time of 2 minutes. We left that to PCR overnight because the machine is heat-regulated so it's fine if it stays in there. </li> |
| </ul><br> | | </ul><br> |
| For tomorrow: check PCR<br> | | For tomorrow: check PCR<br> |
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| <li><dl><dt>Troubleshooting:</dt> | | <li><dl><dt>Troubleshooting:</dt> |
| <dd>template problem: instead of using colony PCR with bacteria, miniprep plasmids first</dd> | | <dd>template problem: instead of using colony PCR with bacteria, miniprep plasmids first</dd> |
- | <dd>PCR conditions: maybe use extension time of 3 minutes instead of 2; maybe lower self annealing temperature from 55 to 52.</dd> | + | <dd>PCR conditions: maybe use extension time of 3 minutes instead of 2; maybe lower self annealing temperature from 55°C to 52°C.</dd> |
| <dd>possible contaminants in sample (unlikely because the TRP1 amplifier worked fine)</dd> | | <dd>possible contaminants in sample (unlikely because the TRP1 amplifier worked fine)</dd> |
| <dd>check sequences of clonat (pAG25) and LEU2</dd> | | <dd>check sequences of clonat (pAG25) and LEU2</dd> |
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| <dd>RC: 1358 bp</dd> | | <dd>RC: 1358 bp</dd> |
| <dd>RL: 2415 bp</dd></dl></li> | | <dd>RL: 2415 bp</dd></dl></li> |
- | <li>Prepared for yeast transformation: made one 250mL bottle of YPD liquid media, colony PCR'ed EST+TRP1, made yeast cultures of W303a and W303alpha. | + | <li>Prepared for yeast transformation: made one 250mL bottle of YPD liquid media, colony PCR'ed EST+TRP1, made yeast cultures of W303a and W303α. |
| <br> | | <br> |
| YPD liquid media: | | YPD liquid media: |
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| <tr><td>peptone (2%)</td><td class="right">5g</td></tr> | | <tr><td>peptone (2%)</td><td class="right">5g</td></tr> |
| <tr><td>glucose (40%)</td><td class="right">12.5 mL</td></tr> | | <tr><td>glucose (40%)</td><td class="right">12.5 mL</td></tr> |
- | <tr><td>distilled H2O</td><td class="right"">237.5 mL</td></tr></table></li> | + | <tr><td>distilled H<sub>2</sub>O</td><td class="right"">237.5 mL</td></tr></table></li> |
| <li><dl><dt>Colony PCR:</dt> | | <li><dl><dt>Colony PCR:</dt> |
- | <dd>took bacteria from pFA6a-TRP1 plasmid bottle and swirled in 200 uL H2O</dd> | + | <dd>took bacteria from pFA6a-TRP1 plasmid bottle and swirled in 200μL H<sub>2</sub>O</dd> |
- | <dd>added 20 uL TRP1, 2.5 uL of EST1 forward and reverse primers into 4 PCR tubes</dd> | + | <dd>added 20μL TRP1, 2.5μL of EST1 forward and reverse primers into 4 PCR tubes</dd> |
| <dd>used PCR program 333</dd></dl></li> | | <dd>used PCR program 333</dd></dl></li> |
| <li><dl><dt>yeast culture:</dt> | | <li><dl><dt>yeast culture:</dt> |
- | <dd>took W303a and W303alpha colonies and added to ~20 mL YPD (2%)</dd> | + | <dd>took W303a and W303α colonies and added to ~20 mL YPD (2%)</dd> |
- | <dd>put samples in shaker (37 degrees, 250 rpm)</dd> | + | <dd>put samples in shaker (37°C, 250 rpm)</dd> |
| <dd>For tomorrow: check PCR, do yeast transformation</dd> | | <dd>For tomorrow: check PCR, do yeast transformation</dd> |
| </dl></li></ul> | | </dl></li></ul> |
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| <ul> | | <ul> |
| <li>Finished another 250mL bottle of liquid YPD media by adding 12.5mL glucose.</li> | | <li>Finished another 250mL bottle of liquid YPD media by adding 12.5mL glucose.</li> |
- | <li>Worked with Lily and Jaeho to take a look at the yeast cultures we inoculated yesterday (W303a and W303alpha). The W303alpha seemed to be contaminated with bacteria, so we started counting the number of cells per mL of yeast in the W303a culture. We used a hemacytometer to count, and determined that if we used that culture to do a yeast transformation, the yield would be very low (ideal number of cells/mL is 5 million, we had around 2.5 million). </li> | + | <li>Worked with Lily and Jaeho to take a look at the yeast cultures we inoculated yesterday (W303a and W303α). The W303α seemed to be contaminated with bacteria, so we started counting the number of cells per mL of yeast in the W303a culture. We used a hemacytometer to count, and determined that if we used that culture to do a yeast transformation, the yield would be very low (ideal number of cells/mL is 5 million, we had around 2.5 million). </li> |
| <li>We abandoned the W303a culture, deciding to do a new culture of the strain, this time from newer plates (the ones we grew yesterday were taken from old plates).</li> | | <li>We abandoned the W303a culture, deciding to do a new culture of the strain, this time from newer plates (the ones we grew yesterday were taken from old plates).</li> |
| <li>Looked at newer colonies of W303a under a microscope, the yeast on the new plates look healthy.</li> | | <li>Looked at newer colonies of W303a under a microscope, the yeast on the new plates look healthy.</li> |
- | <li>Purified PCR product from yesterday (EST1+TRP1) and separated into two 200 uL samples.</li> | + | <li>Purified PCR product from yesterday (EST1+TRP1) and separated into two 200μL samples.</li> |
| <li>After purification, the concentration of each sample was measured using the nanodrop, then stored at -20°C.<br> | | <li>After purification, the concentration of each sample was measured using the nanodrop, then stored at -20°C.<br> |
| <dl><dt>Nanodrop results:</dt> | | <dl><dt>Nanodrop results:</dt> |
- | <dd>EST1+TRP1(1): 81.4 ng/uL</dd> | + | <dd>EST1+TRP1(1): 81.4 ng/μL</dd> |
- | <dd>EST1+TRP1(2): 77.9 ng/uL</dd></dl></li></ul> | + | <dd>EST1+TRP1(2): 77.9 ng/μL</dd></dl></li></ul> |
| For tomorrow: yeast transformation (for real this time)<br> | | For tomorrow: yeast transformation (for real this time)<br> |
| | | |
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| <h3>6/21/14</h3><em>(Nolana)</em> | | <h3>6/21/14</h3><em>(Nolana)</em> |
| <ul> | | <ul> |
- | <li>Looked at newly inoculated yeast cells (W303a, W303alpha) under microscope. The cells were were round and budding, meaning they were healthy and not contaminated.</li> | + | <li>Looked at newly inoculated yeast cells (W303a, W303α) under microscope. The cells were were round and budding, meaning they were healthy and not contaminated.</li> |
- | <li>Worked with Jaeho and Lily to count cells. The original yeast cultures had too many cells to count, so we used a 110uL 1:10 dilution of W303alpha and a 100uL 1:100 dilution of W303a. | + | <li>Worked with Jaeho and Lily to count cells. The original yeast cultures had too many cells to count, so we used a 110μL 1:10 dilution of W303α and a 100μL 1:100 dilution of W303a. |
| <dl><dt>Average number of cells:</dt> | | <dl><dt>Average number of cells:</dt> |
| <dd>W303a: 1,355,000 cells/mL</dd> | | <dd>W303a: 1,355,000 cells/mL</dd> |
- | <dd>W303alpha: 2,020,500 cells/mL</dd></dl></li> | + | <dd>W303α: 2,020,500 cells/mL</dd></dl></li> |
| <li>Calculated ratios of YPD and culture to make a cell density of 5x10<sup>6</sup> cells/mL.</li> | | <li>Calculated ratios of YPD and culture to make a cell density of 5x10<sup>6</sup> cells/mL.</li> |
| <li>Inoculated cultures with YPD and put in shaker at 3pm.<br> | | <li>Inoculated cultures with YPD and put in shaker at 3pm.<br> |
| <dl><dd>W303a: 10.25mL culture + 14.7mL YPD = 25 mL total</dd> | | <dl><dd>W303a: 10.25mL culture + 14.7mL YPD = 25 mL total</dd> |
- | <dd>W303alpha: 9.9mL culture + 4mL YPD = 13.9mL total | + | <dd>W303α: 9.9mL culture + 4mL YPD = 13.9mL total |
| <br><em>Note: total should have been 25mL, but we thought we did not have enough culture. | | <br><em>Note: total should have been 25mL, but we thought we did not have enough culture. |
| But actually, we did the math wrong and had to redo the inoculation, finally putting the cultures into the shaker at 5pm, to be checked at 7:30.</em></dd> | | But actually, we did the math wrong and had to redo the inoculation, finally putting the cultures into the shaker at 5pm, to be checked at 7:30.</em></dd> |
| <dd>W303a: 6.5x10<sup>7</sup> cells/mL ----> 2.5mL culture + 25mL YPD = 27.5 mL total</dd> | | <dd>W303a: 6.5x10<sup>7</sup> cells/mL ----> 2.5mL culture + 25mL YPD = 27.5 mL total</dd> |
- | <dd>W303alpha: 4.5x10<sup>7</sup> cells/mL ----> 2.5mL culture + 22.5mL YPD = 25mL total</dd></dl></li> | + | <dd>W303α: 4.5x10<sup>7</sup> cells/mL ----> 2.5mL culture + 22.5mL YPD = 25mL total</dd></dl></li> |
- | <li>Yeast cells were ready at 8:45pm, with cell density ~2x10^7</li> | + | <li>Yeast cells were ready at 8:45pm, with cell density ~2x10<sup>7</sup></li> |
- | <li>Followed Small-scale LiAc Yeast Transformation Procedure up to step 21, where Jae and I plated the yeast (which had EST1+TRP1 added to them) on TRP- plates, along with two negative controls (W303a and W303alpha without EST1+TRP1). The plates were left in the 30 degree incubator to sit for 2-3 days.</li> | + | <li>Followed Small-scale LiAc Yeast Transformation Procedure up to step 21, where Jae and I plated the yeast (which had EST1+TRP1 added to them) on TRP- plates, along with two negative controls (W303a and W303α without EST1+TRP1). The plates were left in the 30°C incubator to sit for 2-3 days.</li> |
| </ul> | | </ul> |
| | | |
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| <ul> | | <ul> |
| <li>Checked yeast selective plates: there were ~4 colonies per plate, but that was including the negative controls, which should not have any colonies (possible mutation in yeast strain?)</li> | | <li>Checked yeast selective plates: there were ~4 colonies per plate, but that was including the negative controls, which should not have any colonies (possible mutation in yeast strain?)</li> |
- | <li>Worked with Lily to streak out plates for each colony (4 W303a +TRP1 plates, 4 W303a neg. con. plates, 3 W303alpha+TRP1 plates, 4 W303 neg. con. plates), and put all 15 plates in 30 degree incubator. These plates were split into quadrants, where each day ~12:30pm, we will check the colony growth and restreak in the next quadrant. Each quadrant, to be checked daily, goes through ~20 divisions. Hopefully we will see the growth of the colonies fade and come back, following the growth curve of an EST1 knockout.</li> | + | <li>Worked with Lily to streak out plates for each colony (4 W303a +TRP1 plates, 4 W303a neg. con. plates, 3 W303α+TRP1 plates, 4 W303 neg. con. plates), and put all 15 plates in 30° incubator. These plates were split into quadrants, where each day ~12:30pm, we will check the colony growth and restreak in the next quadrant. Each quadrant, to be checked daily, goes through ~20 divisions. Hopefully we will see the growth of the colonies fade and come back, following the growth curve of an EST1 knockout.</li> |
| <li>Poured YPD plates (three 250mL bottles)</li> | | <li>Poured YPD plates (three 250mL bottles)</li> |
- | <li>Colony PCR'ed EST1+LEU2 and RAD52+LEU2: what we changed from the last PCR was the annealing temperature to 54 degrees instead of 55, and the extension time from 2 minutes to 3 minutes, using PCR program 333.</li> | + | <li>Colony PCR'ed EST1+LEU2 and RAD52+LEU2: what we changed from the last PCR was the annealing temperature to 54°C instead of 55°C, and the extension time from 2 minutes to 3 minutes, using PCR program 333.</li> |
| <li>Inoculated plasmids for tomorrow's miniprep (pAG25, pFA6a-TRP1, pFA6a-LEU2MX6, pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP).</li> | | <li>Inoculated plasmids for tomorrow's miniprep (pAG25, pFA6a-TRP1, pFA6a-LEU2MX6, pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP).</li> |
| </ul> | | </ul> |
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| <ul> | | <ul> |
| <li>Worked with Jaeho to miniprep plasmids (pAG25, pFA6a-TRP1, pFA6a-LEU2MX6)</li> | | <li>Worked with Jaeho to miniprep plasmids (pAG25, pFA6a-TRP1, pFA6a-LEU2MX6)</li> |
- | <li>Ran gel of PCR samples (EST1+LEU2 and RAD52+LEU2) on a 0.8% gel. There were no results. Need to check if primer sequences are correct.</li> | + | <li>Ran gel of PCR samples (EST1+LEU2 and RAD52+LEU2) on a 0.8% gel. <br><br> |
| + | <img src=" https://static.igem.org/mediawiki/2014/0/00/CU_625_PCR_Gel_Run_LEU2.jpg " width="350" /> |
| + | <br><br> |
| + | There were no results. Need to check if primer sequences are correct.</li> |
| <li>Nanodropped plasmids from miniprep: | | <li>Nanodropped plasmids from miniprep: |
- | <dl><dd>pAG25 (clonat): 563.7 ng/uL</dd> | + | <dl><dd>pAG25 (clonat): 563.7 ng/μL</dd> |
- | <dd>pFA6a-TRP1: 455.7 ng/uL</dd> | + | <dd>pFA6a-TRP1: 455.7 ng/μL</dd> |
- | <dd>pFA6a-LEU2MX6: 799.7 ng/uL</dd></li> | + | <dd>pFA6a-LEU2MX6: 799.7 ng/μL</dd></li> |
| <li>Checked why PCR gel didn't work: we looked at the primer sequences but there was no problem.</li> | | <li>Checked why PCR gel didn't work: we looked at the primer sequences but there was no problem.</li> |
| <li>We made a 1:1000 dilution of each plasmid (pAG25, pFA6a-TRP1, pFA6a-LEU2MX6), then ran another PCR, this time with the purified plasmids from the miniprep.<br> | | <li>We made a 1:1000 dilution of each plasmid (pAG25, pFA6a-TRP1, pFA6a-LEU2MX6), then ran another PCR, this time with the purified plasmids from the miniprep.<br> |
| <dl> | | <dl> |
- | <dd>5 uL plasmid</dd> | + | <dd>5μL plasmid</dd> |
- | <dd>15 uL H2O</dd> | + | <dd>15μL H<sub>2</sub>O</dd> |
- | <dd>2.5 uL each of the EST1 deletion primers (forward and reverse)</dd> | + | <dd>2.5μL each of the EST1 deletion primers (forward and reverse)</dd> |
| <dd>PCR conditions were the same as last time, program 333.</dd></dl></li></ul> | | <dd>PCR conditions were the same as last time, program 333.</dd></dl></li></ul> |
| <br> | | <br> |
| + | <img src=" https://static.igem.org/mediawiki/2014/8/89/CU_625_W303A_ctrl-1.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/0/06/CU_625_W303A_ctrl-2.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/a/a8/CU_625_W303A_ctrl-4.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/d/d9/CU_625_W303A_TRP1-1.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/2/21/CU_625_W303A_TRP1-2-1.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/b/ba/CU_625_W303A_TRP1-3.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/4/4f/CU_625_W303alpha_ctrl-3.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/e/ea/CU_625_W303alpha_TRP1-1.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/6/66/CU_625_W303alpha_TRP1-2.jpg " width="200" /><br><br> |
| For tomorrow: check PCR and run a gel; check on yeast selective plates. | | For tomorrow: check PCR and run a gel; check on yeast selective plates. |
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| <em>(Jae Ho)</em> | | <em>(Jae Ho)</em> |
| <br> | | <br> |
- | PCR Check Done, Gel run.<br> | + | PCR Check Done, Gel run.<br><br> |
- | 2-3 kbp results for EST1+LEU2, no results on other. Possibly contaminated template of Clonet. <br> | + | <img src=" https://static.igem.org/mediawiki/2014/6/68/CU_626_PCR.jpg " width="350" /><br> |
| + | 2-3 kbp results for EST1+LEU2, no results on other. Possibly contaminated template of Clonet. <br><br> |
| Digest and run gel of clonet to check if it is contaminated etc<br> | | Digest and run gel of clonet to check if it is contaminated etc<br> |
| Yeast cultures are struck and photos taken. <br> | | Yeast cultures are struck and photos taken. <br> |
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| <li>looked up enzymes and buffer for clonat double digest: we will use the enzyme sites SacI and HindIII, and the NEB Cutsmart buffer</li></ul> | | <li>looked up enzymes and buffer for clonat double digest: we will use the enzyme sites SacI and HindIII, and the NEB Cutsmart buffer</li></ul> |
| <br> | | <br> |
| + | <img src=" https://static.igem.org/mediawiki/2014/2/20/CU_626_W303A_Ctrl-1.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/f/f0/CU_626_W303A_TRP1-1.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/8/8f/CU_626_W303A_TRP1-2.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/3/36/CU_626_W303A_TRP1-3.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/e/e2/CU_626_W303alpha_ctrl-1.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/5/5b/CU_626_W303alpha_ctrl-2.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/7/70/CU_626_W303alpha_ctrl-3.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/5/53/CU_626_W303alpha_ctrl-4.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/5/53/CU_626_W303alpha_TRP1-1.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/f/fe/CU_626_W303alpha_TRP1-2.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/b/bb/CU_626_W303alpha_TRP1-4.jpg " width="200" /> |
| + | <img src=" https://static.igem.org/mediawiki/2014/f/f2/CU626_W303alpha_TRP1-3.jpg " width="200" /><br><br> |
| For Monday: run digest<br> | | For Monday: run digest<br> |
| | | |
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| <tr><td colspan="2">Total Volume: 20 uL</td></tr></table></li> | | <tr><td colspan="2">Total Volume: 20 uL</td></tr></table></li> |
| <li>Designed BioBrick primer for EST2 (for yeast strains Professor Medvedik brought back from Boston)</li> | | <li>Designed BioBrick primer for EST2 (for yeast strains Professor Medvedik brought back from Boston)</li> |
- | <li>Streaked 5 YPD plates with yeast strains and put them in 30 degree incubator: W303alpha, 1300, 1294, 1296, 1297</li> | + | <li>Streaked 5 YPD plates with yeast strains and put them in 30 degree incubator: W303α, 1300, 1294, 1296, 1297</li> |
| <li>Helped Wilfrido make two 0.8% gels, and two 1% gels</li> | | <li>Helped Wilfrido make two 0.8% gels, and two 1% gels</li> |
- | <li>Ran a 0.8% gel. Wells: 1. Versa Ladder ; 2. digested clonat ; 3. undigested clonat ; 4-7. Shoshana's samples</li> | + | <li>Ran a 0.8% gel. <br><br> |
| + | <img src=" https://static.igem.org/mediawiki/2014/c/cb/CU_630_Gel_Run.jpg " width="350" /><br> |
| + | Wells: 1. Versa Ladder ; 2. digested clonat ; 3. undigested clonat ; 4-7. Shoshana's samples</li> |
| <li>Gel results: the SacI cut at 43bp, and the BamHI-HF at 1280, which gave two pieces of 1237bp and 2467bp, confirmed by the gel. The undigested clonat result also looked good. This confirmed that the plasmid we have is what we want, the pAG25 (clonat). Now, the question is why didn't it work with the deletion primers?</li> | | <li>Gel results: the SacI cut at 43bp, and the BamHI-HF at 1280, which gave two pieces of 1237bp and 2467bp, confirmed by the gel. The undigested clonat result also looked good. This confirmed that the plasmid we have is what we want, the pAG25 (clonat). Now, the question is why didn't it work with the deletion primers?</li> |
| <li>Made yeast cultures of W303a, 1880, 1882 for yeast transformation tomorrow</li> | | <li>Made yeast cultures of W303a, 1880, 1882 for yeast transformation tomorrow</li> |
| </ul> | | </ul> |
| <br> | | <br> |
- |
| |
| For tomorrow: run PCR of MAK31 and VPS75; yeast transformation<br> | | For tomorrow: run PCR of MAK31 and VPS75; yeast transformation<br> |
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- | <div class="bottom"> | + | <div class="cu-footer"> |
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- | <a href="http://www.cooper.edu" target="_blank"><img src="https://static.igem.org/mediawiki/2014/9/90/CU_2014_Cooper_union_logo.png" height="150" /></a> | + | <img src="https://static.igem.org/mediawiki/2014/b/b2/CU_2014_logoS.png" height="100" style="vertical-align: top;" title="Social" /> |
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