Team:UT Dallas/week4

From 2011.igem.org

(Difference between revisions)
(Created page with "<h2>Week 4</h2> <table> <tr><td valign='top'>1/8/11</td><td valign='top'> Miniprep<br> Observations: i2.11.6 grew, i2.10.7 no growth<br> Making glycerol stock<br> Dissolving and...")
 
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<table>
<table>
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<tr><td valign='top'>1/8/11</td><td valign='top'>
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<tr><td valign='top'>7/25/2011</td><td valign='top'>
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Miniprep<br>
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Observations: i2.11.6 grew, i2.10.7 no growth<br>
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Incubate LB chlora for 1 hour<br>
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Making glycerol stock<br>
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Negative control continued overnight
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Dissolving and diluting primers (10 pmol/ µL)<br>
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Test Experiment #1- Took 10 µL from i2.9.7 and 10 µL from i2.9.8 and mixed them together.<br>Then plated all 20 µL onto a chlora plate.<br>Then we covered it and LED lights in the 37 degrees Celsius incubator and let them grow overnight.
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PCR and gel electrophoresis and purification<br>
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At this stage we have both the FOFr and CheZ* with XbaI and SteI restriction sites.<br>Our next goal will be to insert each part into a vector backbone so they will be ready<br>for addition of the ToxR and Ctx genes. Once these two fusion products are made,<br>we hope to transform and test whether local motion can be inhibited with CheZ and the<br>receptor combo
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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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2/8/11
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</td><td valign='top'>
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Gel electrophoresis and purification<br>
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Dephosphorylation for 1 hour at 37 degrees Celsius, PCR purify<br>
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Ligation at 16 degrees Celsius overnight<br>
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We added both CheZ* and FGFR each into its own vector and if the ligation just performed works we will have 2 new biobricks.<br>The phosphorylation was done to prevent ligase from reannealing the X and S site on the vector.<br>This means only the phosphate on the insert can be used to connect the 2 DNA sequences.
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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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3/8/11
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</td><td valign='top'>
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Transformation of FOFR and CheZ*, DH5a, 50 µL 2 µL DNA at 37 degrees Celsius
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</td></tr><tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr><tr><td valign='top'>
</td></tr><tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr><tr><td valign='top'>
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4/8/11
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7/28/2011
</td><td valign='top'>
</td><td valign='top'>
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Incubation in 3 mL and chlora at 37 degrees Celsius and 220 rpm<br>
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i2.10.1 - FOFR from commercial stock, i2.10.2- negative control, i2.10.3-agar stab being plated on carb plate<br>
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Plating CheZ (K283006) and ToxR (J07009)<br>
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Incubation FOFR colonies (3 mL of LB and carb)<br>
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Dephosphorylation for 1 hour at 37 degrees Celsius, PCR purify
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Plating mutated CheZ (inoculation)<br>
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grew FOFR (fibroblast growth factor receptor) which was on pCMV- SPORT6.1 in E.coli strain DH1OB<br>
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grew FOFR (fibroblast growth factor receptor) which was on pCMV- SPORT6.1 in E.coli strain DH1OB
</td></tr><tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr><tr><td valign='top'>
</td></tr><tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr><tr><td valign='top'>
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5/8/11
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7/29/2011
</td><td valign='top'>
</td><td valign='top'>
Making glycerol stock<br>
Making glycerol stock<br>
Miniprep<br>
Miniprep<br>
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Incubation in 3 mL LB carb at 37 degrees Celsius and 220 rpm overnight<br>
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Observations:<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;i2.10.3 grew, i2.10.1 grew, i2.10.2 didn’t grow<br>
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Test digestion ( 1 hour and 30 minutes)- about 200 ng of DNA<br>
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Sore i2.10.5 until primers come in, then proceed to PCR, gel electrophoresis/purification, digest, gel electrophoresis/purity
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Gel electrophoresis<br>
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Observations: i2.15.16 looks positive, the rest are negative
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</td></tr><tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr><tr><td valign='top'>
</td></tr><tr><td valign='top'>&nbsp;</td><td valign='top'>&nbsp;</td></tr><tr><td valign='top'>
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6/8/11
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7/31/2011
</td><td valign='top'>
</td><td valign='top'>
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Glycerol stock and miniprep</td></tr>
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Incubation of CheZ (I2IT)- 3mL of LB and carb</td></tr>
</table>
</table>

Latest revision as of 01:07, 28 September 2011

Week 4

7/25/2011

Incubate LB chlora for 1 hour
Negative control continued overnight Test Experiment #1- Took 10 µL from i2.9.7 and 10 µL from i2.9.8 and mixed them together.
Then plated all 20 µL onto a chlora plate.
Then we covered it and LED lights in the 37 degrees Celsius incubator and let them grow overnight.

  

7/28/2011

i2.10.1 - FOFR from commercial stock, i2.10.2- negative control, i2.10.3-agar stab being plated on carb plate
Incubation FOFR colonies (3 mL of LB and carb)
Plating mutated CheZ (inoculation)
grew FOFR (fibroblast growth factor receptor) which was on pCMV- SPORT6.1 in E.coli strain DH1OB
grew FOFR (fibroblast growth factor receptor) which was on pCMV- SPORT6.1 in E.coli strain DH1OB

  

7/29/2011

Making glycerol stock
Miniprep
Observations:
     i2.10.3 grew, i2.10.1 grew, i2.10.2 didn’t grow
Sore i2.10.5 until primers come in, then proceed to PCR, gel electrophoresis/purification, digest, gel electrophoresis/purity

  

7/31/2011

Incubation of CheZ (I2IT)- 3mL of LB and carb
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