Team:UT Dallas/week7

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<h2>Week 7</h2>
<h2>Week 7</h2>
<table>
<table>
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<tr><td valign='top'>8/21/11</td><td valign='top'>Incubation: GFP</td></tr>
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<tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr>
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<tr><td valign='top'>8/15/11</td><td valign='top'>
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<tr><td valign='top'>8/22/11</td><td valign='top'>Glycerol Stock/Miniprep GFP<br>
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PCR of ptetR, RBS, LuxI, term<br>
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Test Digestion + Gel Electrophoresis: GFP<br>
+
Gel electrophoresis and purification<br>
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Dissolving/Diluting Primers for ctx promoter and FGFR<br>
+
Glycerol stock and miniprep<br>
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PCR/Gel purification: ctx</td></tr>
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Incubation in 3 mL LB can overnight at 37 degrees Celsius and 220 rpm<br>
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<tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr>
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Test digestion for 1 hour and 30 minutes at 37 degrees Celsius<br>
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<tr><td valign='top'>8/23/11</td><td valign='top'>Full Digestion: CheZ, CheZ*, ctx promoter<br>
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Gel Electrophoresis<br>
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Dissolving/Diluting Primers: -----------------------------<br>
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Test Digestion Observation: <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Lane 5 gave positive results for FGF-R: 2 bands observed at<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; predicted bands size. Lane 4, 7, 9 were all negative bad results<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; because EcoRI was used but only 1 band showed. <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;This suggests that FGFR was not inserted.<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Lanes 10, 11, 12 were CheZ* and all matched negative control. <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Results are bad for CheZ*- will be regrown/miniprep/ligated.<br>
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Gel Electrophoresis/ Purification of CheZ, CheZ*<br>
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Incubation in 3 mL LB chlora overnight at 37 degrees Celsius and 220 rpm
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Ligation: ctx with CheZ, CheZ*</td></tr>
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</td></tr><tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr><tr><td valign='top'>
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<tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr>
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8/16/11
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<tr><td valign='top'>8/24/11</td><td valign='top'>PCR Point Mutation: FGFR (get rid of EcoRI restriction site)<br>
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</td><td valign='top'>
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Transformation (XL10 ultra competent): FGFR (point mutated) <br>
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Digestion; Gel Electrophoresis and Purification<br>
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Transformation: Ctx-CheZ (Carb), ctx-CheZ* (Chlora)</td></tr>
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Ligation (10 minutes at room temperature)<br>
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<tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr>
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Transformation (50 µL DH5a, 2 µL DNA, 37 degrees Celsius overnight, all carb)<br>
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<tr><td valign='top'>8/25/11</td><td valign='top'>Incubation: FGFR
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Re- incubating <br> taking 20 µL of yesterday’s incubations and placing them in 3 mL of LB and appropriate<br>
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Digestion, Gel Electrophoresis/Purification: CheZ, CheZ*<br>
+
Transformation (50 µL DH5a, 2 µL DNA, 37 degrees Celsius overnight, all carb)
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Ligation Overnight: Ctx --> CheZ, CheZ*</td></tr>
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</td></tr><tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr><tr><td valign='top'>
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<tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr>
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8/17/11
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<tr><td valign='top'>8/26/11</td><td valign='top'>Glycerol Stock/Miniprep: FGFR<br>
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</td><td valign='top'>
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Transformation CheZ, CheZ*<br>
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Glycerol stock ToxR and CheZ* colonies (2nd attempt)<br>
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Test Digestion, Gel Electrophoresis<br>
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Miniprep<br>
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Made LbB Chlora & LB Kan</td></tr>
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no results on gel<br>
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<tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr>
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Incubate in 3 mL LB carb overnight at 37 degrees Celsius and 220 rpm<br>
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<tr><td valign='top'>8/27/11</td><td valign='top'>Digestion, Gel Electrophoresis/Purification: RBS-TetR, GFP<br>
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Transformation (50 µL DH5a, 2 µL DNA, 37 degrees Celsius overnight, all carb)<br>
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Ligation (10 min): GFP into RBS-TetR-double term<br>
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Incubation in 3 mL of LB chlora overnight at 37 degrees Celsius and 220 rpm
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Transformation: GFP-RBS-TetR-term</td></tr>
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</td></tr><tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr><tr><td valign='top'>
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8/18/11
 +
</td><td valign='top'>
 +
Glycerol Stock- 350 µL cells and 150 µL 50% glycerol<br>
 +
Miniprep<br>
 +
Test digestion<br>
 +
Digestion, gel electrophoresis and purification<br>
 +
Glycerol stock colonies 4 and 5 CheZ*<br>
 +
Miniprep<br>
 +
Test digestion and gel electrophoresis<br>
 +
Ligation at 16 degrees Celsius overnight<br>
 +
Incubation in 3 mL LB carb overnight at 37 degrees Celsius and 220 rpm<br>
 +
Plate (507011) from Agar Stab
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</td></tr><tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr><tr><td valign='top'>
 +
8/19/11</td><td valign='top'> Transformation (DH5-alpha): Pcst-RBS-LuxI-Term (pSB1C3) -Chlora<br>
 +
Glycerol Stock + Miniprep: pTet+mRFP, RBS-TetR-Term<br>
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Incubation: ctx-GFP (LB Kan)
 +
</td></tr><tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr><tr><td valign='top'>
 +
8/20/11</td><td valign='top'> Transformation: BBa_e0040 GFP Plate 1 Well 14K pSB1A2<br>
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Glycerol Stock/Miniprep: ctx-GFP</td></tr>
</table>
</table>

Latest revision as of 00:55, 28 September 2011

Week 7

8/15/11

PCR of ptetR, RBS, LuxI, term
Gel electrophoresis and purification
Glycerol stock and miniprep
Incubation in 3 mL LB can overnight at 37 degrees Celsius and 220 rpm
Test digestion for 1 hour and 30 minutes at 37 degrees Celsius
Gel Electrophoresis
Test Digestion Observation:
     Lane 5 gave positive results for FGF-R: 2 bands observed at
      predicted bands size. Lane 4, 7, 9 were all negative bad results
      because EcoRI was used but only 1 band showed.
     This suggests that FGFR was not inserted.
      Lanes 10, 11, 12 were CheZ* and all matched negative control.
     Results are bad for CheZ*- will be regrown/miniprep/ligated.
Incubation in 3 mL LB chlora overnight at 37 degrees Celsius and 220 rpm

  

8/16/11

Digestion; Gel Electrophoresis and Purification
Ligation (10 minutes at room temperature)
Transformation (50 µL DH5a, 2 µL DNA, 37 degrees Celsius overnight, all carb)
Re- incubating
taking 20 µL of yesterday’s incubations and placing them in 3 mL of LB and appropriate
Transformation (50 µL DH5a, 2 µL DNA, 37 degrees Celsius overnight, all carb)

  

8/17/11

Glycerol stock ToxR and CheZ* colonies (2nd attempt)
Miniprep
no results on gel
Incubate in 3 mL LB carb overnight at 37 degrees Celsius and 220 rpm
Transformation (50 µL DH5a, 2 µL DNA, 37 degrees Celsius overnight, all carb)
Incubation in 3 mL of LB chlora overnight at 37 degrees Celsius and 220 rpm

  

8/18/11

Glycerol Stock- 350 µL cells and 150 µL 50% glycerol
Miniprep
Test digestion
Digestion, gel electrophoresis and purification
Glycerol stock colonies 4 and 5 CheZ*
Miniprep
Test digestion and gel electrophoresis
Ligation at 16 degrees Celsius overnight
Incubation in 3 mL LB carb overnight at 37 degrees Celsius and 220 rpm
Plate (507011) from Agar Stab

  
8/19/11 Transformation (DH5-alpha): Pcst-RBS-LuxI-Term (pSB1C3) -Chlora

Glycerol Stock + Miniprep: pTet+mRFP, RBS-TetR-Term
Incubation: ctx-GFP (LB Kan)

  
8/20/11 Transformation: BBa_e0040 GFP Plate 1 Well 14K pSB1A2
Glycerol Stock/Miniprep: ctx-GFP