Team:UIUC Illinois/Notebook

From 2014.igem.org

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<h2>Wednesday 11<sup>th</sup> June</h2>
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<h2>Friday 13<sup>th</sup> June</h2>
<a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
<a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
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Followed bacterial transformation protocol to prepare/grow culture overnight<br><br>
Followed bacterial transformation protocol to prepare/grow culture overnight<br><br>
Inoculated 5 mL of M9 with CBB1 and placed it in 30 degree celsius shaker for 3 days<br><br>
Inoculated 5 mL of M9 with CBB1 and placed it in 30 degree celsius shaker for 3 days<br><br>
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<ul style="text-indent:30px;"><li>Previous CBB1 culture did not grow, showed slight growth</li><br><br>
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<ul style="text-indent:30px;"><li>Previous CBB1 culture did not grow, showed slight growth</li>
<li>pDCAF3 strain in M9 did grow in the 30 degree celsius shaker</li></ul><br><br>
<li>pDCAF3 strain in M9 did grow in the 30 degree celsius shaker</li></ul><br><br>
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<h2>Monday 16<sup>th</sup> June</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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No growth in M9 by CBB1<br><br>
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To grow the promoter plasmid:<br><br>
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<ul style="text-indent:30px;">
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<li>5 mL of LB + 1 uL Amp</li>
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<!--something sameer has to translate-->
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<li>Label tubes</li>
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<li>Grow in 30 degree celsius shaker overnight</li>
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</ul><br><br>
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New CBB1 trials:<br><br>
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<ul style="text-indent:30px;"><li>2 tubes of 1 mL M9 + 2 tubes of 1 mL LB each with 10 uL of sample</li></ul><br><br>
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Purify genomic DNA plasmid following the Wizard Protocol<br><br>
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<ul style="text-indent:30px;">
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<li>Added lysis buffer before centrifuging. So we separated (translate) sample into 2 tubes and will (translate) accordingly</li>
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<li>We put our 2 promoter plasmid plates in the 4 degree celsius shelf</li>
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</ul><br><br>
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<h2>Tuesday 17<sup>th</sup> June</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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Quantified gDNA with TECAN<br><br>
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<ul style="text-indent:30px;"><li>Result: 5.4 ng/uL</li></ul><br><br>
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Made 2 plates of M9 culture and 2 plates of CBB1 culture (100 uL used) then placed them in 37 degree celsius shaker<br><br>
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<h2>Wednesday 18<sup>th</sup> June</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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Autoclaved 50% glycerol<br><br>
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Made four 25% glycerol stock solutions (stored in the -80 degree celsius freezer):<br><br>
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<ul style="text-indent:30px;">
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  }
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<li>CBB1 in M9 (x2 1.5 mL tubes)</li>
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<li>Promoter 1 </li>
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display:none;
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<li>Promoter 2 (both promoter plasmids are J23114 Ampᴿ)</li>
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}
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</ul><br><br>
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</style>
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Purified both promoter plasmids using the purification protocol<br><br>
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<ul style="text-indent:30px;"><li>Stored in the -20 degree celsius freezer in the DNA box</li></ul><br><br>
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{{:Team:UIUC_Illinois/Footer}}
{{:Team:UIUC_Illinois/Footer}}

Revision as of 02:00, 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


The Institute for Genomic Biology
Biosystems Design, South wing, 2nd floor
University of Illinois at Urbana-Champaign
1206 West Gregory Drive
61801 Urbana, Illinois
illinoisigem@gmail.com
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