Team:UIUC Illinois/Notebook

From 2014.igem.org

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<html><div id="Wednesday_9th_July.html"></div></html>
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<div class ="tbnote">
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<h2>Wednesday 9<sup>th</sup> July</h2>
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<a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
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<div style="clear: both;"></div>
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Made 400mL of M9 solution
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<ul style="text-indent:30px;">
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<li>Made 40% glucose solution of 50mL: 2g glucose</li>
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<li>Added the following to 316mL of DI H2O: Na2HPO4: 13.56g Na2HPO4, 2g NH4Cl, 1g NaCl, 6g KH2PO4</li>
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<li>Added 80mL 5x M9 salt solution (20% of 400mL)</li>
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<li>Added 800uL MgSO4 + 40uL CaCl2</li>
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<li>Added 4mL glucose solution using syringe</li>
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<li>Stirred until all dissolved</li>
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</ul><br><br>
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Removed competent cells from the -80 freezer<br><br>
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Thawed on ice for 30 minutes<br><br>
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Removed CAM agar plate from 4 degree cold room<br><br>
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Mixed 4uL of pDCAF3 plasmid with 30uL competent cells in microcentrifuge tube, placed on ice for 30 minutes<br><br>
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Placed mixture in water bath (42C) for 45 seconds<br><br>
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Placed mixture on ice for 2 minutes<br><br>
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Added 300uL LB to mixture<br><br>
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Placed in 37 degree shaker for 45 minutes<br><br>
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Plated cells on CAM plate, placed in 37 degree drawer overnight<br><br>
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<html><div id="Thursday_10th_July.html"></div></html>
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<html>
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<div class ="tbnote">
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<h2>Thursday 10<sup>th</sup> July</h2>
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<a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
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<div style="clear: both;"></div>
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GuaB/pDCAF suspension in caffeine/theobromine
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<ul style="text-indent:30px;">
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<li>0.0975g/10mL caffeine, 0.09g/10mL -> 50 mM</li>
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<li>2.5mL of M9 (with Glucose) + 2.4875mL of H2O + 12.5uL of caffeine/theobromine</li>
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<li>Use syringe to filter out 10mL of caffeine/theobromine into new conical test tubes</li>
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<li>Take out guaB/pDCAF from -80C freezer, suspend it using 10uL pipette tips</li>
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<li>Place them into 37C inclubator overnight</li>
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</ul><br><br>
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</div>
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</html>
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<html><div id="Tuesday_15th_July.html"></div></html>
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<html>
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<div class ="tbnote">
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<h2>Tuesday 15<sup>th</sup> July</h2>
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<a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
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<div style="clear: both;"></div>
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Retrieved lactobacillus WCFS from Dr. Lu<br><br>
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</div>
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</html>
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<html><div id="Wednesday_16th_July.html"></div></html>
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<html>
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<div class ="tbnote">
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<h2>Wednesday 16<sup>th</sup> July</h2>
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<a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
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<div style="clear: both;"></div>
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Incubate 4 tubes with LB+CM
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<ul style="text-indent:30px;">
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<li>Red 1 and 2</li>
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<li>Green 1 and 2</li>
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<li>Placed in shaker at 12:20pm</li>
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</ul><br><br>
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</div>
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</html>
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Revision as of 20:27, 17 October 2014


Monday 9th June

Made LB broth using 10 grams of bacto agar + 12 grams HiVeg LB broth + 600 mL ddH2O

Autoclaved 1.5 mL Eppendorf tubes as well as LB

Poured plates of LB and chloramphenicol (CAM)

Streaked 2 plates with E. coli DguaB (pDCAF3)

Incubated in 37 degrees celsius

Aliquoted kanamycin (KAN) in four 1mL tubes

Tuesday 10th June

Autoclaved at 1:30pm

  • 0.8 grams of YNB + 156 mL H2O
  • 28 grams of salts + 490 mL H2O

Used sterile filter for 1M MgSO4 and 1M CaCl2

Suspended 5 uL of CM34 (antibiotic) + 5 mL of LB + 1 colony of pDCAF3 E. coli

Added .5 grams of caffeine + 400 uL of 1M MgSO4 + 20 uL 1M CaCl2 + 40 mL M95x salts + H2O to YNB + H2O mixture

  • Labeled this "M9 media"

Inoculated frozen stock of E. coli guaB pDCAF3 in 5 mL of M9 media

Placed sample in 30 degree celsius shaker for culturing

Wednesday 11th June

Inoculated CBB1 frozen stock in 5 mL of M9 media

Placed sample in 30 degree celsius shaker

Purified pDCAF plasmid using DNA mini kit

  • Plasmid concentration was 132.9 ng/uL


Friday 13th June

Added 10 uL deionized H2O into the J23114 promoter plasmid (Ampᴿ)

Followed bacterial transformation protocol to prepare/grow culture overnight

Inoculated 5 mL of M9 with CBB1 and placed it in 30 degree celsius shaker for 3 days

  • Previous CBB1 culture did not grow, showed slight growth
  • pDCAF3 strain in M9 did grow in the 30 degree celsius shaker


Monday 16th June

No growth in M9 by CBB1

To grow the promoter plasmid:

  • 5 mL of LB + 1 uL Amp
  • Label tubes
  • Grow in 30 degree celsius shaker overnight
New CBB1 trials:

  • 2 tubes of 1 mL M9 + 2 tubes of 1 mL LB each with 10 uL of sample


Purify genomic DNA plasmid following the Wizard Protocol

  • Added lysis buffer before centrifuging. So we separated (translate) sample into 2 tubes and will (translate) accordingly
  • We put our 2 promoter plasmid plates in the 4 degree celsius shelf


Tuesday 17th June

Quantified gDNA with TECAN
  • Result: 5.4 ng/uL


Made 2 plates of M9 culture and 2 plates of CBB1 culture (100 uL used) then placed them in 37 degree celsius shaker

Wednesday 18th June

Autoclaved 50% glycerol

Made four 25% glycerol stock solutions (stored in the -80 degree celsius freezer):

  • CBB1 in M9 (x2 1.5 mL tubes)
  • Promoter 1
  • Promoter 2 (both promoter plasmids are J23114 Ampᴿ)
Purified both promoter plasmids using the purification protocol
  • Stored in the -20 degree celsius freezer in the DNA box


Thursday 19th June

Quantified promoter plasmid (J23114 #1 and #2)
    #1: 342.9 ng/ul with a ratio of 1.9 #2: 373.2 ng/ul with a ratio of 1.91


Checking to see if expired enzymes work: Using the J23114 promoter plasmid, we will see if the 3-year expired SpeI works
  • We combined 1 uL of 2.1 buffer, 1 uL of Spe1, and 6 uL of J23114 promoter
  • Heat bath at 37 degrees celsius for 1 hour because 2.1 buffer is <100% effective on this enzyme
  • Then we will run gel electrophoresis: Concluded that the enzyme works


Cutting the promoter with restriction enzymes
  • Combined 24 uL of J23114 promoter + 3 uL of CutSmart buffer + 1 uL of each EcoRI and SpeI
  • Heat bath at 37 degrees celsius for 1 hour + 20 minute heat-kill at 80 degrees celsius


Cutting linearized backbone
  • Linearized backbone with chloraphenicol marker (12 uL) + 12 uL water + 1 uL of each EcoRI, PstI, and DpnI + 3 uL 2.1 buffer
  • Heat bath at 37 degrees celsius for 1 hour + 20 minute heat-kill at 80 degrees celsius


Wednesday 9th July

Made 400mL of M9 solution
  • Made 40% glucose solution of 50mL: 2g glucose
  • Added the following to 316mL of DI H2O: Na2HPO4: 13.56g Na2HPO4, 2g NH4Cl, 1g NaCl, 6g KH2PO4
  • Added 80mL 5x M9 salt solution (20% of 400mL)
  • Added 800uL MgSO4 + 40uL CaCl2
  • Added 4mL glucose solution using syringe
  • Stirred until all dissolved


Removed competent cells from the -80 freezer

Thawed on ice for 30 minutes

Removed CAM agar plate from 4 degree cold room

Mixed 4uL of pDCAF3 plasmid with 30uL competent cells in microcentrifuge tube, placed on ice for 30 minutes

Placed mixture in water bath (42C) for 45 seconds

Placed mixture on ice for 2 minutes

Added 300uL LB to mixture

Placed in 37 degree shaker for 45 minutes

Plated cells on CAM plate, placed in 37 degree drawer overnight

Thursday 10th July

GuaB/pDCAF suspension in caffeine/theobromine
  • 0.0975g/10mL caffeine, 0.09g/10mL -> 50 mM
  • 2.5mL of M9 (with Glucose) + 2.4875mL of H2O + 12.5uL of caffeine/theobromine
  • Use syringe to filter out 10mL of caffeine/theobromine into new conical test tubes
  • Take out guaB/pDCAF from -80C freezer, suspend it using 10uL pipette tips
  • Place them into 37C inclubator overnight


Tuesday 15th July

Retrieved lactobacillus WCFS from Dr. Lu

Wednesday 16th July

Incubate 4 tubes with LB+CM
  • Red 1 and 2
  • Green 1 and 2
  • Placed in shaker at 12:20pm