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            <p> <div class  = "dateheader"> 6.04.15 </div> </p>
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<font color = "orange"><b><u>Goal:</u></b></font> Streaked out plates, continue competent cell protocol.
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<li>Streaked out MACH cells, Top10, and Biosensor Estrogen - on to LB plates. The biosensor estrogen negative plates had kanamyacin antibiotic on it (10 ul + 50 ul water).</li>
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<li>Began TOP10 Competent cell protocol (protocol <a href = "#">here</a>).
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<div class = "indentlist"><ul>Diluted TOP10 overnight culture 1:100 for a 2ml volume in LB. Grew it from 10a-5p. Then added 300 ul to each 500 ml of LB in sterile 2L flasks. Placed in 18C shaker overnight after removing the magnetic stir rod.</ul></div> <!-- indentlist -->
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     <p> <div class  = "weekheader"> Week 3 </div> </p>
 
     <p> <div class  = "weekheader"> Week 3 </div> </p>

Revision as of 17:57, 7 August 2015

Under Construction.

The CMU iGEM team did a lot this summer from building a light to playing with an Arduino to improving an estrogen sensor. Luckily, we tracked everything in our handy-dandy notebook.

"I have successfully inserted content into the first tab"

Week 1

Take me to Tab 2

May 26th, 2015
  • First day of work!
  • Familiarized ourselves with the lab.
  • Made LB (protocol) & poured plates (protocol).

The light fluoresces! We have transformants & isolated proteins.

Week 1

5.28.15
Goal: To do Miniprep to purify the DNA. It is a small scale isolation of plasmid DNA from bacteria. Then do restriction enzyme digest on plasmid DNA and gel electrophoresis. This technique can be used for cleaving DNA molecules at specific sites, ensuring that all DNA fragments that contain a particular sequence have the same size; furthermore, each fragment that contains the desired sequence has the sequence located at exactly the same position within the fragment. The cleavage method makes use of enzymes isolated from bacteria called restriction enzymes. They are able to cleave DNA molecules at positions at which particular short sequences of bases are present.
  • Did MiniPreps of MACH cells expressing fluorescence to isolate plasmid DNA from MACH cells (protocol here)
  • Performed restriction enzyme digestion on J23108, J23109, J23111 constructs
      For promoters, PST1 & Spe1 were used. For insert, xba1 and Pst1 were used.
  • Ran an Agarose Gel Electrophoresis
  • Cut digests from gels
For next time: Will prepare Colony PCR to determine if we got the correct restriction sites and sequences from the given interlab devices.

Week 2

6.01.15

Goal: To submit minipreps for sequencing, will get results in two days and compare our constructions.
  • Did minipreps on samples where the devices were incorporated in (J promoters & I3504)<.li>
  • Send minipreps off for sequencing.
      5 µL water, 5 µL sample, 1 µL of each primer

6.02.15

Goal: Make competent MACH cells.
  • Made competitent MACH cells (protocol here)
  • measured fluorescence of GFP for 6 samples
      108A, 108C, 109A, 109C, 111A, 111B
For next time: Since we did it without MACH cells, will redo tomorrow with MACH cells and biosensor to compare results.

6.03.15

Goal: Observe Sequence Data and compare to our own plasmids. Redo TECAN results with blank and biosensor to see differences in fluorescent data.
  • Compared sequence data to registry/our own plasmids. Figured out that no promoters got inserted in. Decided to send in Minipreps of the parts of the original devices for sequencing to see if the given parts were what we expected.
  • Redid fluorescence reading
      Very evident difference, shows the presence of GFP
      May redo in the future in order to do the trianalysis.

6.04.15

Goal: Streaked out plates, continue competent cell protocol.
  • Streaked out MACH cells, Top10, and Biosensor Estrogen - on to LB plates. The biosensor estrogen negative plates had kanamyacin antibiotic on it (10 ul + 50 ul water).
  • Began TOP10 Competent cell protocol (protocol here).
      Diluted TOP10 overnight culture 1:100 for a 2ml volume in LB. Grew it from 10a-5p. Then added 300 ul to each 500 ml of LB in sterile 2L flasks. Placed in 18C shaker overnight after removing the magnetic stir rod.

Week 3

Week 4

Week 5

Estrogen Sensor:

  • tried newly transformed cells, all taken from the same plate
  • tried to get similar-sized colonies
  • tested the following amounts of estrogen:
    • 100 uM, 20 uM, 10 uM, 1 uM, 100 nM, 10 nM, 1 nM, 0 nM
  • got very varied and inconsistent results
  • no pattern between amount of estrogen added & reading
  • high standard deviation
  • think that there is a problem with the transformation
  • will try another transformation

yo this is cool