Difference between revisions of "Team:BostonU/Notebook"

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<h2 <!--style="padding: 5px; background-color: #990000; font-family: Calibri; color: #FFFFFF; font-size: 30px;"-->Notebook</h2>
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<h3>Notebook</h3>
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<h4>May</h4>
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<table style="background-color: #F0F0F0" width="100%" border="0" cellspacing="0" cellpadding="0">
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<h5>Week One - May 28th to May 29th</h5>
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<ul>
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<tr>
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<li>May 28th 2015
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<td colspan="2" scope="col"><h2 >May</h2></td>
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<ol>
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</tr>
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<li>Made broth and agar</li>
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<li>Plated the agar</li>
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<tr>
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<li>Aliquot 50mL LB media tubes</li>
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<td colspan="2" scope="col"><h3>Week of May 18</h3></td>
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<li>Innoculate Ben’s mammalian backbones with dimerization domains and Ben’s phiC-31, TP901-1, ofr7 and gp3 plasmids</li>
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</tr>
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</ol>
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</li>
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<tr>
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<li>May 29th 2015
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        <td colspan="2" scope="col">
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<ol>
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• Made broth and agar. <br>
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<li>Cell stock creation from frozen plasmids</li>
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• Plated the agar.<br>
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<li>Miniprepped the plasmids we grew up and cell stocked for iGEM cell stocks</li>
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• Allocated media into 50mL portions to avoid contamination of an entire suppply<br>
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<li>Using the nanodrop fluorospectrometer</li>
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• Innoculated protein plasmids from frozen stock.<br>
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</ol>
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• Created cell stocks from frozen plasmids.<br>
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</li>
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• Miniprepped the cell stocks to isolate the desired plasmids<br>
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</ul>
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•  Used the nanodrop flourospectrometer to determine the concentrations of our plasmids
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<h5>Week Two - June 1st to June 5th</h5>
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</td>
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<ul>
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      </tr>
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<li>June 1st 2015
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<ol>
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<tr>
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<li>Digested Ben's backbones</li>
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<td colspan="2" scope="col"><h3>Week of May 25</h3></td>
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<li>PCR set-up</li>
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</tr>
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<li>Made an agarose gel to separate the products of the PCR</li>
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<li>Made more pS-5 and pS-18 cell stocks (Our pS-5 and pS-18 cell stocks gave us a very low yield when their concentrations were tested with a nanodropper).</li>
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<tr>
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</ol>
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<td colspan="2" scope="col">
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</li>
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<li>June 2nd 2015
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•  Digested the backbone plasmids were miniprepped
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<ol>
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    <br />
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<li>Mini-prepped the pS-5 and pS-18 cell stocks that were made yesterday. </li>
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    •  Set up PCR reactions to create split DNA sequences of the proteins we'll be using
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<li>Measured the concentrations of DNA from the purified digestion results and purified gel results. </li>
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    <br />
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<li>Digested pS-5 because it is a backbone. pS-18 is not a backbone that is why it was not re-digested. </li>
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    •  Made an agarose gel and ran gel electrophoresis on the PCR results
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<li>Ran the PCR for pS-14 and 10 different primers for a second time, the first time we did this was yesterday but the test yielded strange results in that our bands were thick, clouded, and our ladder was relatively useless because it did not run for a long enough period of time. </li>
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    <br />
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<li>Ran the gel for the results from today’s PCR in order to see if we can get better results than yesterday and we did.</li>
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    •  Purified results from gel
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<li>Gel purified</li>
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    <br />
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<li>Digesting Our Two Integrases with Enzymes in Order to Ligate (the pS-14 phi 31 integrases into pS-1:12 backbones)</li>
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    •  Also created new cell stocks of plasmids that had low yields when originally minprepped
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<li> PCR Purification of Our Restriction Results to Get the Integrase Inserts</li>
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<br />
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<li>Ligation</li>
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    •  Miniprepped the new cell stocks
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</ol>
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    <br />
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</li>
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    •  Measured concentrations of DNA from purified gel and miniprep
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    <br />
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    •  Digested backbones from miniprep in order to yield sticky ends for ligation of protein split into the plasmid
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−
    <br />
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    •  Gel electrophoresis from PCR reactions yielded strange results so we performed the PCR reactions again
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    <br />
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    •  Ran gel with new PCR results
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−
    <br />
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    •  Performed gel purification to isolate DNA
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−
    <br />
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    •  Digested and purified our inserts to complement the sticky ends we created in our backbones
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    <br />
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    •  Ligated our inserts into the backbones and transformed them into top10 cells
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    <br />
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    •  Plated the transformed cells to grow colonies
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    <br />
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    •  Checked the colonies after they incubated overnight, a couple had low yields<br />
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•  Picked three colonies of each ligation and placed them in deepwells to incubate overnight
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    <br />
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    •  Removed the deepwells from incubator, spun them down in the centrifuge and removed the media.
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<br />
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    •  Miniprepped the cells from each deepwell to isolate the plasmid that contains both the insert and backbone.<br />
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&nbsp;•  Digested a small amount of DNA from each ligation to perform a test cut. After digestion we ran the DNA through a gel. The desired result was two clear bands, one that was the size of the backbone plasmid and one was the insert. The actual results were not ideal so we decided to retrace our steps.</li>
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</td>
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</tr>
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<br>
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<tr><td colspan="2" scope="col"><h2>June</h2></td></tr>
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<tr><td colspan="2" scope="col"><h3>Week of June 1</h3></td></tr>
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<tr><td colspan="2" scope="col">
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• Picked new colonies from ligations that failed to show promising test cuts.<br />
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• We also performed new PCR reactions on inserts that didn't look promising initially. We changed the settings on the thermocycler and also adjusted the amount of primers we were using per reaction.
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    <br />
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    • Ran a new gel to see the results of the PCR reaction<br />
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• Performed gel purification of inserts that were the proper length
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    <br />
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    • We performed a new set of ligations which failed, since no colonies grew after the top10 cells containing the ligated plasmid were plated. </td>
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</tr>
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<tr><td colspan="2" scope="col"><h3>Week of June 8</h3></td></tr>
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<tr><td colspan="2" scope="col">
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    </td></tr>
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<tr><td colspan="2" scope="col"><h3>Week of June 15</h3></td></tr>
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<tr><td colspan="2" scope="col">
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    </td>
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    <tr><td colspan="2" scope="col"><h3>Week of June 22</h3></td></tr>
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<tr><td colspan="2" scope="col">
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    </td>
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</tr>
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    <tr><td colspan="2" scope="col"><h3>Week of June 29</h3></td></tr>
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<tr><td colspan="2" scope="col">
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    </td>
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    </tr>
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Revision as of 21:33, 17 September 2015

Our Project
Day by Day Protocols

Notebook

May

Week One - May 28th to May 29th
  • May 28th 2015
    1. Made broth and agar
    2. Plated the agar
    3. Aliquot 50mL LB media tubes
    4. Innoculate Ben’s mammalian backbones with dimerization domains and Ben’s phiC-31, TP901-1, ofr7 and gp3 plasmids
  • May 29th 2015
    1. Cell stock creation from frozen plasmids
    2. Miniprepped the plasmids we grew up and cell stocked for iGEM cell stocks
    3. Using the nanodrop fluorospectrometer
Week Two - June 1st to June 5th
  • June 1st 2015
    1. Digested Ben's backbones
    2. PCR set-up
    3. Made an agarose gel to separate the products of the PCR
    4. Made more pS-5 and pS-18 cell stocks (Our pS-5 and pS-18 cell stocks gave us a very low yield when their concentrations were tested with a nanodropper).
  • June 2nd 2015
    1. Mini-prepped the pS-5 and pS-18 cell stocks that were made yesterday.
    2. Measured the concentrations of DNA from the purified digestion results and purified gel results.
    3. Digested pS-5 because it is a backbone. pS-18 is not a backbone that is why it was not re-digested.
    4. Ran the PCR for pS-14 and 10 different primers for a second time, the first time we did this was yesterday but the test yielded strange results in that our bands were thick, clouded, and our ladder was relatively useless because it did not run for a long enough period of time.
    5. Ran the gel for the results from today’s PCR in order to see if we can get better results than yesterday and we did.
    6. Gel purified
    7. Digesting Our Two Integrases with Enzymes in Order to Ligate (the pS-14 phi 31 integrases into pS-1:12 backbones)
    8. PCR Purification of Our Restriction Results to Get the Integrase Inserts
    9. Ligation